Molecular genetic approach to treatment and diagnosis of alcohol and drug dependence

ABSTRACT

Compositions and methods are provided that are useful for diagnosing, treating, and monitoring alcohol dependence and disorders, susceptibility to alcohol dependence disorders, as well as drug related dependence and disorders. The methods include treating patients with an antagonist of the serotonin receptor 5-HT3 for such disorders, wherein the patient&#39;s serotonin transporter gene SLC6A4 is known to have particular genotypes.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.16/784,051, filed on Feb. 6, 2020, which is a continuation of U.S.patent application Ser. No. 15/397,076, filed on Jan. 3, 2017, which isa continuation of U.S. patent application Ser. No. 14/266,313, filed onApr. 30, 2014, which is a divisional of U.S. patent application Ser. No.13/589,603, filed on Aug. 20, 2012, which is a continuation under 35U.S.C. § 111(a) of and claims the benefit of priority to InternationalPatent Application Serial No. PCT/US2011/042823, filed on Jul. 1, 2011,and published on Jan. 5, 2012 as WO 2012/003462 and republished on Mar.22, 2012, which is entitled to priority pursuant to 35 U.S.C. § 119(e)to U.S. provisional patent application No. 61/361,203, filed on Jul. 2,2010; 61/429,416, filed on Jan. 3, 2011; and 61/488,328, filed on May20, 2011. The entire disclosures of the afore-mentioned patentapplications are incorporated herein by reference.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with government support under Grant Nos.AA010522-12, AA0032903, AA001016 and AA012964 awarded by the NationalInstitutes of Health. The government has certain rights in theinvention.

BACKGROUND

Alcohol abuse and dependence are widespread and it is estimated that 14million American adults abused alcohol or were dependent on it in 1992and that approximately 10% of Americans will be affected by alcoholdependence sometime during their lives. Alcohol dependence,characterized by the preoccupation with alcohol use, tolerance, andwithdrawal, is a chronic disorder with genetic, psychosocial, andenvironmental factors influencing its development and manifestations.Studies have demonstrated the significance of opioids (i.e.,beta-endorphin), dopamine (DA), serotonin (5-HT), γ-amino-butyric acid(GABA) and glutamate for the development and maintenance of alcoholdependence.

Various medications and behavioral therapy have been used to treatalcohol dependence. The neuronal targets of alcohol include manyneurotransmitter systems and the molecules participating in orregulating the systems, including GABA, glutamate, DA, opioids, andserotonin (for a review see Johnson, 2004, Expert Opin. Pharmacother.,5:9:1943-1955).

Despite the number of studies performed in this area, few drugs foralcohol dependence are approved in the U.S. The approved drugs aredisulfiram, naltrexone, Vivitrex®/Vivitrol® (a long-acting depotformulation of naltrexone), and acamprosate. Disulfiram is anirreversible inhibitor of aldehyde dehydrogenase leading to increasedlevels of acetaldehyde, a toxic intermediate in alcohol metabolism.Patients who take disulfiram and drink alcohol experience an increaseddilation of arterial and capillary tone producing hypotension, nausea,vomiting, flushing, headache and possibly in some, worse symptoms.Therefore, the concept behind the use of disulfiram is that thealcohol-dependent individual associates drinking with unpleasant adverseevents and, as a result, avoids further alcohol consumption.Nevertheless, recent research shows that disulfiram has limited utilitybecause compliance is low unless it is administered by a partner orspouse.

Serotonin (5-HT) dysfunction probably contributes to the development ofalcoholism. Serotonin's receptors contribute to alcohol use in animals,as alcohol increases basal levels of 5-HT affecting receptors. Of theseven distinct families of 5-HT receptors, three are known to contributeto alcohol dependence: 5-HT_(1A) receptors might be associated withalcohol consumption and the development of tolerance; 5-HT₂ receptorswith reward; and 5-HT₃ receptors with the development of reinforcement.Based on such evidence, several serotonergic drugs have been examined,but with inconsistent results. Presently only sertraline and ondansetron(a serotonin-3 (5-HT₃) antagonist) appear to show any promise withcertain subtypes of alcoholic patients and fluoxetine with depressedalcoholics (see Kenna, 2005, Drug Discovery Today: TherapeuticStrategies, 2:1:71-78 and Johnson, 2000, Alcohol. Clin. Exp. Res.,24:1597-1601).

The 5-HT₃ receptor is involved in the expression of alcohol's rewardingeffects. Behavioral pharmacological studies show that many of alcohol'srewarding effects are mediated by interactions between DA and 5-HTreceptors in the midbrain and cortex. 5-HT receptors are denselydistributed in the terminals of mesocorticolimbic DA containing neurons,where they regulate DA release in these brain regions. These DApathways, particularly those in the NAc, are involved in mediating therewarding effects of abused substances including alcohol. Demonstrationthat 5-HT₃ receptor blockade reduces DA activity, and therefore therewarding effects of abused drugs (including alcohol), comes from atleast three different animal paradigms. 5-HT₃ receptor antagonists: 1)attenuate hyperlocomotion in the rat induced by DA or ethanol injectioninto the nucleus accumbens; 2) inhibit DiMe-C7 (a neurokinin)—inducedhyperlocomotion, which is also attenuated by the DA antagonist,fluphenazine; and 3) decrease alcohol consumption in several animalmodels and across different species.

Animal studies demonstrated that the 5-HT₃ receptor facilitates some ofthe biochemical and behavioral effects of alcohol through midbrain DArelease. 5-HT₃ antagonists are consistently shown to suppress alcoholpreference in animal studies, with recent evidence suggesting the5-HT_(3A) receptor subunit requisite for 5-HT₃ antagonist-inducedreductions in alcohol consumption.

Ondansetron, a 5-HT₃ receptor antagonist, has functionally oppositeeffects to SSRIs and blocks serotonin agonism at the 5-HT₃ receptor.According to studies, ondansetron can be effective for early-onsetalcoholics (EOA) but not late-onset alcoholics (LOA), where age of onsetof alcoholism (younger versus older than 25 years old) is the basis forsubtyping alcoholics (Johnson, 2000, Alcohol. Clin. Exp. Res.,24:1597-1601). In a placebo-controlled trial, 271 participants werestratified into EOA and LOA subtypes by 1, 4, and 16 μg/kg twice-dailydoses of ondansetron compared with placebo (Johnson, 2000, J. Am. Med.Assoc., 284:963-971). Patients with EOA who received ondansetron showedsignificant reductions in drinking (particularly those receiving 4 μg/kgtwice daily) compared with LOA across all groups. In another study, itwas shown that ondansetron treatment is more likely to be associatedwith improved drinking outcomes among EOA compared with LOA (Kranzler etal., (2003, Alcohol. Clin. Exp. Res., 27:1150-1155). Ondansetroncontinues to be examined for individuals with early-onset alcoholism.

The reasons for these differential effects are unknown; however, onehypothesis suggests that alcoholics with a biological predispositionhave a dysregulation of serotonergic function primarily associated withserotonin transporter (SERT) function (Johnson, 2000, Alcohol. Clin.Exp. Res. 24:1597-1601). The polymorphic variation of the SERT (the5′-HTTLPR) is hypothesized to be involved with the effectiveness ofondansetron and sertraline in EOA and LOA alcohol-dependent individuals,respectively. Given that epidemiologic studies demonstrate that alcoholdependence has an approximately 50-60% heritability, the prospect forpositive outcomes to drug therapy at least partly dependent on geneticpredisposition in some alcoholics is strong. Recent studies have,therefore, attempted to delineate the genetic components associated withalcohol dependence. These findings highlight the role that 5-HT plays inalcohol consumption, although drug trials using serotonergics have haddifficulty delineating responders from non-responders.

Vulnerability to alcohol dependence is heritable, with a rate rangingfrom 0.52 to 0.64 (Kendler, 2001). Despite this high heritability rate,only one marker allele (alcohol-metabolizing aldehyde dehydrogenasegenes) has been identified consistently to be associated with alcoholism(Kranzler et al, 2002). Of the various neurotransmitter systems throughwhich alcohol mediates its effects, the serotonergic system has beenshown to play a role in alcohol preference and consumption (Johnson,2004). Synaptic serotonergic neurotransmission is terminated whenserotonin (5-HT) is transported back into pre-synaptic neurons by 5-HTtransporters (5-HTTs) (Talvenheimo and Rudnick, 1980). Therefore, amajor part of the functional capacity of the serotonergic system isregulated by the 5-HTT. Heavy episodic drinking is associated withnumerous psychiatric and general medical conditions causing a majorpublic health burden (Cargiulo, 2007). Several studies have reported adose-response relationship between the extent of heavy drinking and therisk of alcohol related morbidity and mortality among heavy drinkers(Makela and Mustonen, 2007; Gastfriend et al., 2007). Consequently,reduction of heavy drinking is used as an indicator of treatmentresponse in clinical trials aimed at treating alcohol dependence.

There is a long felt need in the art for compositions and methods usefulfor diagnosing, treating, and monitoring alcohol disorders andsusceptibility to alcohol disorders.

SUMMARY OF THE INVENTION

The present invention relates to molecular genetics techniques topredict which alcohol or drug dependent subjects are amenable tospecific treatments and to predict those subjects for which suchtreatment might produce an adverse event.

The present invention also relates to methods and assays useful fordetermining whether a subject has a predisposition to developing anaddictive disease or disorder, determining whether a subject will beresponsive to particular treatments, and compositions and methods usefulfor treating a subject in need of treatment.

The present invention also relates to compositions and methods usefulfor treating subjects having an addictive disease or disorder (or whoare predisposed thereto) based on identification of genetic markersindicative of a subject being predisposed to such disease or disorder orbeing predisposed to responding to treatment thereof.

The present invention also relates to molecular genetics techniquesand/or other ways to subtype groups by biological or psychologicalmeasures or variables to determine which subjects will respond best totreatment for an addictive disease or disorder.

These and other aspects that will become apparent are based on thediscovery that molecular genetics techniques can be used to predictwhich alcohol or drug dependent subjects are amenable to specifictreatments and to predict those subjects for which such treatment mightproduce an adverse event. Various aspects of the invention are describedin further detail below.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 provides data showing Log Drinks/Drinking Day in rs1042173 SNPs(i.e., TT vs TG/GG) among 278 alcoholics who received either ondansetronor placebo. Footnote: The mean numbers of Drinks/drinking day values(DDD) represented by “Natural log (X+1)” on the Y axis are as follows:1=1.718; 1.5=3.482; 2=6.389; 2.5=11.182: X=Number of Drinks/drinkingday. Mean DDD in ondansetron (OND) recipients are represented in closedbars, and mean DDD in placebo recipients are represented in open bars;blue and black bars represent TG/GG and TT genotypes respectively.Numbers of subjects in each rs1042173 genotypic group are as follows:TG/GGplacebo-92, TTplacebo-47, TG/GG ond-94 and TTond-45.

FIG. 2 shows the locations 2 SNPs in the 5HT3b gene (top panel),rs4938056 and rs17614942 and 2 SNPs in the 5HT3a gene (bottom panel),rs1150226 and rs1062613.

FIG. 3 shows the DDD in genotypic group rs4938056.

FIG. 4 shows the DDD in genotypic group rs17614942.

FIG. 5 shows the retention rate versus study week in VA/NIDA Study#1025-Topiramate for the Treatment of Methamphetamine Dependence.

FIG. 6 shows the percentage of subjects with a negative methamphetamineuse weeks in weeks 6-12 (all urine drug screens for methamphetamine arenegative).

FIG. 7 shows the percentage of subjects with a negative methamphetamineuse week in weeks 6-12 for light methamphetamine users only (<=18 daysuse) in VA/NIDA Study #1025-Topiramate for the Treatment ofMethamphetamine Dependence.

FIG. 8 shows the percentage of subjects with a negative methamphetamineuse week in weeks 6-12 for heavy methamphetamine users only (>18 daysuse)) VA/NIDA Study #1025-Topiramate for the Treatment ofMethamphetamine Dependence.

FIG. 9 shows the Study Week; Treatment group and last urine result priorto randomization breakdown for the percentage of subjects with anegative methamphetamine use week in weeks 6-12 in VA/NIDA Study#1025—Topiramate for the Treatment of Methamphetamine Dependence.

FIG. 10 shows the probability of the age of onset of methamphetamine usefor each of the three 5′-HTTLPR genotypes.

FIGS. 11A-11C provide data demonstrating that LL/t+ carriers show aneffect on PHDD, DDD and PDA and are responsive to ondansetron treatment.

FIG. 12 depicts data regarding patients with less than 3 (1/month) heavydrinking days (“safe drinking”) during 12 weeks.

DETAILED DESCRIPTION

Abbreviations, Generic Names, and Acronyms

5-HT—serotonin

5-HT₃—a subtype of serotonin receptor, the serotonin-3 receptor

5-HTOL—5-hydroxytryptophol

5-HTT—serotonin transporter (also referred to as SERT, 5HTT, HTT, andOCD1)

5-HTTLPR—serotonin transporter-linked polymorphic region

ADE—alcohol deprivation effect

ADI—adolescence diagnostic interview

ASPD—antisocial personality disorder

AUD—alcohol use disorder

BBCET—Brief Behavioral Compliance Enhancement Treatment

BED—binge eating disorder

b.i.d.—twice a day

B_(max)—maximum specific paroxetine binding density

BRENDA—Biopsychosocial, Report, Empathy, Needs, Direct advice, andAssessment

CBI—combined behavioral intervention

CBT—Cognitive Behavioral Coping Skills Therapy, also referred to ascognitive behavioral therapy

CDT—carbohydrate-deficient transferrin

ChIPS—children's interview for psychiatric syndrome

CMDA—cortico-mesolimbic dopamine

DA—dopamine

DDD—drinks/drinking day

DSM—Diagnostic and Statistical Manual of Mental Disorders

EOA—early-onset alcoholic(s)

G2651T—a site within a putative polyadenylation signal for a commonlyused 3′ polyadenylation site of the SLC6A4 gene; also has referenceidentification number rs1042173 at the GenBank website of the NationalCenter for Biotechnology Information

GABA—γ-amino-butyric acid (also referred to as γ-amino butyric acid andγ-aminobutyric acid)

GGT—γ-glutamyl transferase

ICD—impulse control disorder

IP—intraperitoneal

K_(d)—affinity constant

K_(m)—equilibrium constant

L—long

LOA-late-onset alcoholic(s)

MET—Motivational Enhancement Therapy

miRNA—micro RNA

MM—Medical Management

NAc—nucleus accumbens

Naltrexone—a opioid receptor antagonist

ncRNA—non-coding RNA

NMDA—N-methyl-D-aspartate

NOS—not otherwise specified

Ondansetron (Zofran®)—a serotonin receptor antagonist

P—alcohol-preferring rats

S—short

SERT—serotonin transporter (also referred to as 5-HTT)

SLC6A4—human 5-HT transporter gene.

SNP—single nucleotide polymorphism

SSRI—selective serotonin re-uptake inhibitor

Topiramate (Topamax®)—an anticonvulsant

TSF—Twelve-Step Facilitation Therapy (e.g., Alcoholics Anonymous)

V_(max)—maximum serotonin uptake velocity

VTA—ventral tegmental area

Definitions

In describing and claiming the invention, the following terminology willbe used in accordance with the definitions set forth below. Unlessdefined otherwise, all technical and scientific terms used herein havethe same meaning as commonly understood by one of ordinary skill in theart to which this invention belongs. Any methods and materials similaror equivalent to those described herein can be used in the practice ortesting of the present invention. As used herein, each of the followingterms has the meaning associated with it in this section. Specificvalues listed below for radicals, substituents, and ranges are forillustration only; they do not exclude other defined values or othervalues within defined ranges for the radicals and substituents.

As used herein, the articles “a” and “an” refer to one or to more thanone, i.e., to at least one, of the grammatical object of the article. Byway of example, “an element” means one element or more than one element.

The term “about,” as used herein, means approximately, in the region of,roughly, or around. When the term “about” is used in conjunction with anumerical range, it modifies that range by extending the boundariesabove and below the numerical values set forth. In general, the term“about” is used herein to modify a numerical value above and below thestated value by a variance of 20%.

One of ordinary skill in the art will appreciate that addictivedisorders such as those related to alcohol or drugs, does mean that asubject is dependent unless specifically defined as such.

The term “additional therapeutically active compound,” in the context ofthe present invention, refers to the use or administration of a compoundfor an additional therapeutic use other than just the particulardisorder being treated. Such a compound, for example, could include onebeing used to treat an unrelated disease or disorder, or a disease ordisorder which may not be responsive to the primary treatment for theaddictive disease or disorder being treated. Disease and disorders beingtreated by the additional therapeutically active agent include, forexample, hypertension and diabetes.

As used herein, the term “aerosol” refers to suspension in the air. Inparticular, aerosol refers to the particlization or atomization of aformulation of the invention and its suspension in the air.

Cells or tissue are “affected” by a disease or disorder if the cells ortissue have an altered phenotype relative to the same cells or tissue ina subject not afflicted with a disease, condition, or disorder.

As used herein, an “agonist” is a composition of matter that, whenadministered to a mammal, such as a human, enhances or extends abiological activity of interest. Such effect may be direct or indirect.

The term “alcohol abuser,” as used herein, refers to a subject who meetsDSM IV criteria for alcohol abuse (i.e., “repeated use despite recurrentadverse consequences”) but is not dependent on alcohol.

As used herein, an “analog” of a chemical compound is a compound that,by way of example, resembles another in structure but is not necessarilyan isomer (e.g., 5-fluorouracil is an analog of thymine).

An “antagonist” is a composition of matter that when administered to amammal, such as a human, inhibits or impedes a biological activityattributable to the level or presence of an endogenous compound in themammal. Such effect may be direct or indirect.

As used herein, the term “anti-alcohol agent” refers to any active drug,formulation, or method that exhibits activity to treat or prevent one ormore symptom(s) of alcohol addiction, alcohol abuse, alcoholintoxication, and/or alcohol withdrawal, including drugs, formulationsand methods that significantly reduce, limit, or prevent alcoholconsumption in mammalian subjects.

The term “appetite suppression,” as used herein, is a reduction, adecrease or, in cases of excessive food consumption, an amelioration inappetite. This suppression reduces the desire or craving for food.Appetite suppression can result in weight loss or weight control asdesired.

The term “average drinking,” as used herein, refers to the mean numberof drinks consumed during a one week period. The term “average drinking”is used interchangeably herein with the term “average level ofdrinking.”

A “biomarker” is a specific biochemical in the body which has aparticular molecular feature that makes it useful for measuring theprogress of disease or the effects of treatment, or for measuring aprocess of interest.

A “compound,” as used herein, refers to any type of substance or agentthat is commonly considered a drug, or a candidate for use as a drug, aswell as combinations and mixtures of the above.

A “control” subject is a subject having the same characteristics as atest subject, such as a similar type of dependence, etc. The controlsubject may, for example, be examined at precisely or nearly the sametime the test subject is being treated or examined. The control subjectmay also, for example, be examined at a time distant from the time atwhich the test subject is examined, and the results of the examinationof the control subject may be recorded so that the recorded results maybe compared with results obtained by examination of a test subject.

A “test” subject is a subject being treated.

As used herein, a “derivative” of a compound refers to a chemicalcompound that may be produced from another compound of similar structurein one or more steps, as in replacement of H by an alkyl, acyl, or aminogroup.

As used herein, the term “diagnosis” refers to detecting a risk orpropensity to an addictive related disease or disorder. In any method ofdiagnosis exist false positives and false negatives. Any one method ofdiagnosis does not provide 100% accuracy.

A “disease” is a state of health of a subject wherein the subject cannotmaintain homeostasis, and wherein if the disease is not ameliorated thenthe subject's health continues to deteriorate. In contrast, a “disorder”in a subject is a state of health in which the subject is able tomaintain homeostasis, but in which the subject's state of health is lessfavorable than it would be in the absence of the disorder. However, thedefinitions of “disease” and “disorder” as described above are not meantto supersede the definitions or common usage related to specificaddictive diseases or disorders.

A disease, condition, or disorder is “alleviated” if the severity of asymptom of the disease or disorder, the frequency with which such asymptom is experienced by a patient, or both, are reduced.

As used herein, an “effective amount” means an amount sufficient toproduce a selected effect, such as alleviating symptoms of a disease ordisorder. In the context of administering two or more compounds, theamount of each compound, when administered in combination with anothercompound(s), may be different from when that compound is administeredalone. The term “more effective” means that the selected effect isalleviated to a greater extent by one treatment relative to the secondtreatment to which it is being compared.

The term “elixir,” as used herein, refers in general to a clear,sweetened, alcohol-containing, usually hydroalcoholic liquid containingflavoring substances and sometimes active medicinal agents.

The term “excessive drinker,” as used herein, refers to men who drinkmore than 21 alcohol units per week and women who consume more than 14alcohol units per week. One standard drink is 0.5 oz of absolutealcohol, equivalent to 10 oz of beer, 4 oz of wine, or 1 oz of 100-proofliquor. These individuals are not dependent on alcohol but may or maynot meet DSM IV criteria for alcohol abuse.

As used herein, a “functional” molecule is a molecule in a form in whichit exhibits a property or activity by which it is characterized. Afunctional enzyme, for example, is one that exhibits the characteristiccatalytic activity by which the enzyme is characterized.

The term “heavy drinker,” as used herein, refers to men who drink morethan 14 alcohol units per week and women who consume more than 7 alcoholunits per week. One standard drink is 0.5 oz of absolute alcohol,equivalent to 10 oz of beer, 4 oz of wine, or 1 oz of 100-proof liquor.These individuals are not dependent on alcohol but may or may not meetDSM IV criteria for alcohol abuse.

The term “heavy drinking”, as used with respect to the alcohol-dependentpopulation of Example 1, refers to drinking at least about 21 standarddrinks/week for women and at least 30 drinks/week for men during the 90days prior to enrollment in the study and is more fully describedtherein.

A “heavy drinking day,” as used herein, refers to the consumption by aman or woman of more than about five or four standard drinks perdrinking day, respectively.

The term “heavy drug use,” as used herein, refers to the use of any drugof abuse, including, but not limited to, cocaine, methamphetamine, otherstimulants, phencyclidine, other hallucinogens, marijuana, sedatives,tranquilizers, hypnotics, opiates at intervals or in quantities greaterthan the norm. The intervals of use include intervals such as at leastonce a month, at least once a week, and at least once a day. “Heavy druguse” is defined as testing “positive” for the use of that drug on atleast 2 occasions in any given week with at least 2 days between testingoccasions.

As used herein, the term “inhaler” refers both to devices for nasal andpulmonary administration of a drug, e.g., in solution, powder and thelike. For example, the term “inhaler” is intended to encompass apropellant driven inhaler, such as is used to administer antihistaminefor acute asthma attacks, and plastic spray bottles, such as are used toadminister decongestants.

The term “inhibit a complex,” as used herein, refers to inhibiting theformation of a complex or interaction of two or more proteins, as wellas inhibiting the function or activity of the complex. The term alsoencompasses disrupting a formed complex. However, the term does notimply that each and every one of these functions must be inhibited atthe same time.

As used herein, an “instructional material” includes a publication, arecording, a diagram, or any other medium of expression which can beused to communicate the usefulness of a compound of the invention in thekit for effecting alleviation of the various diseases or disordersrecited herein. Optionally, or alternately, the instructional materialmay describe one or more methods of alleviating the diseases ordisorders in a subject. The instructional material of the kit of theinvention may, for example, be affixed to a container which contains theidentified compound invention or be shipped together with a containerwhich contains the identified compound. Alternatively, the instructionalmaterial may be shipped separately from the container with the intentionthat the instructional material and the compound be used cooperativelyby the recipient.

“Intensity of drinking” refers to the number of drinks, which can beequated with values such as drinks/day, drinks/drinking day, etc.Therefore, greater intensity of drinking means more drinks/day, ordrinks/drinking day, etc.

As used herein, a “ligand” is a compound that specifically binds to atarget compound or molecule. A ligand “specifically binds to” or “isspecifically reactive with” a compound when the ligand functions in abinding reaction which is determinative of the presence of the compoundin a sample of heterogeneous compounds.

A “receptor” is a compound or molecule that specifically binds to aligand.

The term “measuring the level of expression” or “determining the levelof expression” as used herein refers to any measure or assay which canbe used to correlate the results of the assay with the level ofexpression of a gene or protein of interest. Such assays includemeasuring the level of mRNA, protein levels, etc. and can be performedby assays such as northern and western blot analyses, binding assays,immunoblots, etc. The level of expression can include rates ofexpression and can be measured in terms of the actual amount of an mRNAor protein present.

The term “nasal administration” in all its grammatical forms refers toadministration of at least one compound of the invention through thenasal mucous membrane to the bloodstream for systemic delivery of atleast one compound of the invention. The advantages of nasaladministration for delivery are that it does not require injection usinga syringe and needle, it avoids necrosis that can accompanyintramuscular administration of drugs, and trans-mucosal administrationof a drug is highly amenable to self administration.

“Obesity” is commonly referred to as a condition of increased bodyweight due to excessive fat. Drugs to treat obesity are generallydivided into three groups: (1) those that decrease food intake, such asdrugs that interfere with monoamine receptors, such as noradrenergicreceptors, serotonin receptors, dopamine receptors, and histaminereceptors; (2) those that increase metabolism; and (3) those thatincrease thermogenesis or decrease fat absorption by inhibitingpancreatic lipase (Bray, 2000, Nutrition, 16:953-960 and Leonhardt etal., 1999, Eur. J. Nutr., 38:1-13). Obesity has been defined in terms ofbody mass index (BMI). BMI is calculated as weight (kg)/[height (m)]²,according to the guidelines of the U.S. Centers for Disease Control andPrevention (CDC), and the World Health Organization (WHO). Physicalstatus: The use and interpretation of anthropometry. Geneva,Switzerland: World Health Organization 1995. WHO Technical ReportSeries), for adults over 20 years old, BMI falls into one of thesecategories: below 18.5 is considered underweight, 18.5-24.9 isconsidered normal, 25.0-29.9 is considered overweight, and 30.0 andabove is considered obese.

The term “per application” as used herein refers to administration of adrug or compound to a subject.

As used herein, the term “pharmaceutically acceptable carrier” includesany of the standard pharmaceutical carriers, such as a phosphatebuffered saline solution, water, emulsions such as an oil/water orwater/oil emulsion, and various types of wetting agents. The term alsoencompasses any of the agents approved by a regulatory agency of the USFederal government or listed in the US Pharmacopeia for use in animals,including humans.

As used herein, the term “physiologically acceptable” ester or saltmeans an ester or salt form of the active ingredient which is compatiblewith any other ingredients of the pharmaceutical composition, and whichis not deleterious to the subject to which the composition is to beadministered.

A “predisposition” to an addictive disease or disorder refers tosituations in which a subject has an increased chance of abusing asubstance such as alcohol or a drug or becoming addicted to alcohol or adrug or other addictive diseases or disorders.

The term “prevent,” as used herein, means to stop something fromhappening, or taking advance measures against something possible orprobable from happening. In the context of medicine, “prevention”generally refers to action taken to decrease the chance of getting adisease or condition.

The term “problem drinker,” as used herein, encompasses individuals whodrink excessively and who report that their alcohol consumption iscausing them problems. Such problems include, for example, driving whileintoxicated, problems at work caused by excessive drinking, andrelationship problems caused by excessive drinking by the subject.

The term “psychosocial management program,” as used herein, relates tothe use of various types of counseling and management techniques used tosupplement the combination pharmacotherapy treatment of addictive andalcohol-related diseases and disorders.

“Reduce”—see “inhibit”.

The term “reduction in drinking”, as used herein, refers to a decreasein drinking according to one or more of the measurements of drinkingsuch as heavy drinking, number of drinks/day, number of drinks/drinkingday, etc.

The term “regulate” refers to either stimulating or inhibiting afunction or activity of interest.

A “sample,” as used herein, refers to a biological sample from asubject, including, but not limited to, normal tissue samples, diseasedtissue samples, biopsies, blood, saliva, feces, semen, tears, and urine.A sample can also be any other source of material obtained from asubject which contains cells, tissues, or fluid of interest asinterpreted in the context of the claim and the type of assay to beperformed using that sample.

By “small interfering RNAs (siRNAs)” is meant, inter alia, an isolateddsRNA molecule comprising both a sense and an anti-sense strand. In oneaspect, it is greater than 10 nucleotides in length. siRNA also refersto a single transcript that has both the sense and complementaryantisense sequences from the target gene, e.g., a hairpin. siRNA furtherincludes any form of dsRNA (proteolytically cleaved products of largerdsRNA, partially purified RNA, essentially pure RNA, synthetic RNA,recombinantly produced RNA) as well as altered RNA that differs fromnaturally occurring RNA by the addition, deletion, substitution, and/oralteration of one or more nucleotides.

By the term “specifically binds,” as used herein, is meant a moleculewhich recognizes and binds a specific molecule, but does notsubstantially recognize or bind other molecules in a sample, or it meansbinding between two or more molecules as in part of a cellularregulatory process, where the molecules do not substantially recognizeor bind other molecules in a sample.

The term “standard,” as used herein, refers to something used forcomparison. For example, it can be a known standard agent or compoundwhich is administered or added and used for comparing results whenadding a test compound, or it can be a standard parameter or functionwhich is measured to obtain a control value when measuring an effect ofan agent or compound on a parameter or function. Standard can also referto an “internal standard,” such as an agent or compound which is addedat known amounts to a sample and is useful in determining such things aspurification or recovery rates when a sample is processed or subjectedto purification or extraction procedures before a marker of interest ismeasured. Internal standards are often a purified marker of interestwhich has been labeled, such as with a radioactive isotope, allowing itto be distinguished from an endogenous marker.

The term “one standard drink,” as used herein, is 0.5 oz of absolutealcohol, equivalent to 10 oz of beer, 4 oz of wine, or 1 oz of 100-proofliquor.

A “subject” of diagnosis or treatment is a mammal, including a human.

The term “subject comprises a predisposition to the early onset ofalcoholism,” as used herein, refers to a subject who has, or ischaracterized by, a predisposition to the early onset of alcoholism.

The term “symptom,” as used herein, refers to any morbid phenomenon ordeparture from the normal in structure, function, or sensation,experienced by the patient and indicative of disease. In contrast, asign is objective evidence of disease. For example, a bloody nose is asign. It is evident to the patient, doctor, nurse and other observers.

As used herein, the term “treating” may include prophylaxis of thespecific disease, disorder, or condition, or alleviation of the symptomsassociated with a specific disease, disorder or condition and/orpreventing or eliminating the symptoms. A “prophylactic” treatment is atreatment administered to a subject who does not exhibit signs of adisease or exhibits only early signs of the disease for the purpose ofdecreasing the risk of developing pathology associated with the disease.“Treating” is used interchangeably with “treatment” herein.

A “therapeutic” treatment is a treatment administered to a subject whoexhibits signs of pathology for the purpose of diminishing oreliminating those signs.

A “therapeutically effective amount” of a compound is that amount ofcompound which is sufficient to provide a beneficial effect to thesubject to which the compound is administered.

The term “pharmaceutically-acceptable salt” refers to salts which retainthe biological effectiveness and properties of the compounds of thepresent invention and which are not biologically or otherwiseundesirable. In many cases, the compounds of the present invention arecapable of forming acid and/or base salts by virtue of the presence ofamino and/or carboxyl groups or groups similar thereto.

Embodiments

In an aspect, the present invention provides a method of identifyingaddictive disease or disorder patients with favorable prognosis forpharmacological treatment, comprising: identifying genetic patternsfavorable to treatment response.

Another aspect provides a method of identifying addictive disease ordisorder patients with favorable prognosis for pharmacological treatment(the patient can be treated/can respond favorably to treatment),comprising: a) obtaining genetic patterns (genetic variations) of two ormore genetic regions (for example, genetic regions in genes that governserotonin function, including genes that are associated with changes inserotonin transporter function and expression) of the patients, whereinthe genetic patterns are predictive of addictive diseases or disorders;b) standardizing, with a processor (such as a CPU (central processingunit)/processor in a computer), the genetic patterns for each of thegenetic regions, wherein the standardizing, comprises: mapping thepossible range of genetic patterns to a range of conditionalprobabilities ranging from about 0 to about 1; and, c) operating, with aprocessor, upon the standardized genetic patterns using a computationalprocedure to transform the standardized genetic patterns into acomposite result that has a diagnostic or predictive error foridentifying addictive disease or disorder patients with favorableprognosis for pharmacological treatment lower than any of the individualgenetic regions alone.

The polymorphisms identified herein also include their related miRNA,mRNA, ncRNA, or protein expression, levels, or states of function, orother biochemical products or chemical associations, which may serve asbiomarkers.

In another aspect, the present invention provides a method of treatingan addictive disease or disorder, comprising: administering to a patientin need thereof a therapeutically effective amount of an antagonist ofthe serotonin receptor 5-HT₃, wherein the patient's serotonintransporter gene SLC6A4 is known to have:

-   -   (a) 3 or fewer of the genotypes selected from (Set (a)):        -   i. the LS or SS genotype of the functional polymorphism            serotonin transporter-linked polymorphic region 5-HTTLPR;        -   ii. the TG or GG genotype of the single nucleotide            polymorphism (SNP) rs1042173;        -   iii. the GG genotype of the SNP rs1176713;        -   iv. the AA genotype of the SNP rs1176719; and,        -   v. the GG genotype of the SNP rs1672717;    -   (b) 3 or more of the genotypes selected from (Set (b)):        -   i. the LL genotype of the functional polymorphism serotonin            transporter-linked polymorphic region 5-HTTLPR;        -   ii. the AG or GG genotype of SNP rs1176719;        -   iii. the GG or AG genotype of SNP rs1672717 (HTR3B); and,        -   iv. the AA genotype of SNP rs2276307 (HTR3B);    -   (c) 3 or more of the genotypes selected from (Set (c)):        -   i. the LL genotype of the functional polymorphism serotonin            transporter-linked polymorphic region 5-HTTLPR;        -   ii. the TT genotype of rs1042173 (SERT);        -   iii. the GT or GG genotype of rs10160548 (HTR3A);        -   iv. the GA or GG genotype of rs1176746 (HTR3B); and,        -   v. the GG genotype of rs12270070 (HTR3B).    -   (d) a genotype selected from (Set (d)):        -   i. the AG genotype of rs1150226;        -   ii. the AG genotype of rs1150226 and the AC genotype of            rs17614942;        -   iii. the AG genotype of rs1150226, the LL genotype of            5-HTTLPR, and the TT genotype of rs1042173;        -   iv. the AG genotype of rs1150226, the AC genotype of            rs17614942, the LL genotype of 5-HTTLPR, and the TT genotype            of rs1042173;        -   v. the AC genotype of rs17614942; or        -   vi. the AC genotype of rs17614942, the LL genotype of            5-HTTLPR, and the TT genotype of rs1042173;    -   (e) the AA genotype of rs1176719;    -   (f) the GG genotype of rs1176713;    -   (g) the AC genotype of rs17614942 and the LL genotype of        5-HTTLPR;    -   (h) the AG genotype of rs1150226 and at least one genotype        selected from:        -   i. the AC genotype of rs17614942; and        -   ii. the LL genotype of 5-HTTLPR;    -   (i) the AA genotype of rs1176719 and at least one genotype        selected from:        -   i. the AC genotype of rs17614942;        -   ii. the LL genotype of 5-HTTLPR; and,        -   iii. the TT genotype of rs1042173;    -   (j) the AC genotype of rs17614942 and at least one genotype        selected from:        -   i. the AA genotype of rs1176719;        -   ii. the LL genotype of 5-HTTLPR; and,        -   iii. the TT genotype of rs1042173;    -   (k) the GG genotype of rs1176713 and at least one genotype        selected from:        -   i. the LL genotype of 5-HTTLPR; and,        -   ii. the TT genotype of rs1042173;    -   (l) the GG genotype of rs1176713 and at least one genotype        selected from:        -   i. the AC genotype of rs17614942; and,        -   ii. the LL genotype of 5-HTTLPR;    -   (m) the GG genotype of rs1176713 and at least one genotype        selected from:        -   i. the AC genotype of rs17614942; and,        -   ii. the TT genotype of rs1042173;    -   (n) the GG genotype of rs1176713 and at least one genotype        selected from:        -   i. the AG genotype of rs1150226; and,        -   ii. the TT genotype of rs1042173;    -   (o) the TT genotype of rs3758987 and at least one genotype        selected from genotype sets (i)-(iv);        -   i. the TT genotype of rs1042173;        -   ii. the AA genotype of rs2276307 and the LL genotype of            5-HTTLPR;        -   iii. the TT genotype of rs1042173 and the LL genotype of            5-HTTLPR; or        -   iv. the TT genotype of rs1042173, the AA genotype of            rs2276307, and the LL genotype of 5-HTTLPR; or    -   (p) the TC genotype of rs3758987 and at least one genotype        selected from genotype sets (i)-(iv);        -   i. the TT genotype of rs1042173;        -   ii. the AA genotype of rs2276307 and the LL genotype of            5-HTTLPR;        -   iii. the TT genotype of rs1042173 and the LL genotype of            5-HTTLPR; or        -   iv. the TT genotype of rs1042173, the AA genotype of            rs2276307, and the LL genotype of 5-HTTLPR.

In another aspect, the patient is known to have 2 or fewer genotypes(i)-(v) of Set (a).

In another aspect, the patient is known to have 1 or 0 genotypes (i)-(v)of Set (a).

In another aspect, the patient is known to have 0 genotypes (i)-(v) ofSet (a).

In another aspect, the patient is known to have at least three ofgenotypes (i)-(iv) of Set (b).

In another aspect, the patient is known to have all 4 genotypes (i)-(iv)of Set (b).

In another aspect, the patient is known to have at least three ofgenotypes (i)-(v) of Set (c).

In another aspect, the patient is known to have 4 or 5 genotypes (i)-(v)of Set (c).

In another aspect, the patient is known to have 4 genotypes (i)-(v) ofSet (c).

In another aspect, the patient is known to have all 5 genotypes (i)-(v)of Set (c).

In another aspect, the patient is known to have a genotype of Set (d).

In another aspect, the patient is known to have genotype (i) of Set (d).

In another aspect, the patient is known to have genotype (ii) of Set(d).

In another aspect, the patient is known to have genotype (iii) of Set(d).

In another aspect, the patient is known to have genotype (iv) of Set(d).

In another aspect, the patient is known to have genotype (v) of Set (d).

In another aspect, the patient is known to have genotype (vi) of Set(d).

In another aspect, the patient is known to have genotype Set (e).

In another aspect, the patient is known to have genotype Set (f).

In another aspect, the patient is known to have genotype Set (g).

In another aspect, the patient is known to satisfy genotype Set (h).

In another aspect, the patient is known to have genotype (i) of Set (h).

In another aspect, the patient is known to have genotype (ii) of Set(h).

In another aspect, the patient is known to have genotypes (i)-(ii) ofSet (h).

In another aspect, the patient is known to satisfy genotype Set (i).

In another aspect, the patient is known to have at least two ofgenotypes (i)-(iii) of Set (i).

In another aspect, the patient is known to have all three genotypes(i)-(iii) of Set (i).

In another aspect, the patient is known to have genotype (i) of Set (i).

In another aspect, the patient is known to have genotype (ii) of Set(i).

In another aspect, the patient is known to have genotype (iii) of Set(i).

In another aspect, the patient is known to satisfy genotype Set (j).

In another aspect, the patient is known to have at least two ofgenotypes (i)-(iii) of Set ( ).

In another aspect, the patient is known to have all three genotypes(i)-(iii) of Set (j).

In another aspect, the patient is known to have genotype (i) of Set (j).

In another aspect, the patient is known to have genotype (ii) of Set(j).

In another aspect, the patient is known to have genotype (iii) of Set(j).

In another aspect, the patient is known to satisfy genotype Set (k).

In another aspect, the patient is known to have genotype (i) of Set (k).

In another aspect, the patient is known to have genotype (ii) of Set(k).

In another aspect, the patient is known to have genotypes (i)-(ii) ofSet (k).

In another aspect, the patient is known to satisfy genotype Set (1).

In another aspect, the patient is known to have genotype (i) of Set (1).

In another aspect, the patient is known to have genotype (ii) of Set(1).

In another aspect, the patient is known to have genotypes (i)-(ii) ofSet (1).

In another aspect, the patient is known to satisfy genotype Set (m).

In another aspect, the patient is known to have genotype (i) of Set (m).

In another aspect, the patient is known to have genotype (ii) of Set(m).

In another aspect, the patient is known to have genotypes (i)-(ii) ofSet (m).

In another aspect, the patient is known to satisfy genotype Set (n).

In another aspect, the patient is known to have genotype (i) of Set (n).

In another aspect, the patient is known to have genotype (ii) of Set(n).

In another aspect, the patient is known to have genotypes (i)-(ii) ofSet (n).

The method of claim 1, wherein the patient is known to satisfy genotypeset (o).

In another aspect, the patient is known to have at least two ofgenotypes (i)-(iv) of Set (o).

In another aspect, the patient is known to have at least three ofgenotypes (i)-(iv) of Set (o).

In another aspect, the patient is known to have all four of genotypes(i)-(iv) of Set (o).

In another aspect, the patient is known to satisfy genotype Set (p).

In another aspect, the patient is known to have at least two ofgenotypes (i)-(iv) of Set (p).

In another aspect, the patient is known to have at least three ofgenotypes (i)-(iv) of Set (p).

In another aspect, the patient is known to have all four of genotypes(i)-(iv) of Set (p).

In another aspect, the patient is known to have the LL and TT genotypes,and/or the AG genotype of rs1150226 and/or the AC genotype ofrs17614942.

In another aspect, the patient is known to have the LL and TT genotypes,and/or the AG genotype of rs1150226, and/or the AC genotype ofrs17614942, but not the GG genotype of rs1150226.

In another aspect, the patient is known to have the LL and TT genotypes,and/or the AG genotype of rs1150226, and/or the AC genotype ofrs17614942, but not the TG genotype of rs1042173.

In another aspect, the patient is known to have the LL and TT genotypes,and/or the AG genotype of rs1150226, and/or the AC genotype ofrs17614942, but not the GG genotype of rs1150226 or the TG genotype ofrs1042173.

In another aspect, the patient is known to have the LL and TT genotypes,and/or the A+ genotype of rs1150226, and/or the AC genotype ofrs17614942.

In another aspect, the patient is known to have the LL and TT genotypes,and/or the A+ genotype of rs1150226, and/or the AC genotype ofrs17614942, but not the GG genotype of rs1150226.

In another aspect, the patient is known to have the LL and TT genotypes,and/or the A+ genotype of rs1150226, and/or the AC genotype ofrs17614942, but not the TG genotype of rs1042173.

In another aspect, the patient is known to have the LL and TT genotypes,and/or the A+ genotype of rs1150226, and/or the AC genotype ofrs17614942, but not the GG genotype of rs1150226 or the TG genotype ofrs1042173.

In another aspect, the patient is known to have the LL and TT genotypesand/or the AC genotype of rs17614942.

In another aspect, the patient is known to have the LL and TT genotypesand/or the AG genotype of rs1150226 and the AC genotype of rs17614942.

In another aspect, the patient is known to have the AG genotype ofrs1150226 and/or the AC genotype of rs17614942.

Examples of the antagonist of the serotonin receptor 5-HT₃ includeondansetron, tropisetron, granisetron, palonosetron, dolasetron, andmetocclopromide.

In another aspect, the antagonist of the serotonin receptor 5-HT₃ isondansetron.

Examples of the dosage of the antagonist of the serotonin receptor 5-HT₃(e.g., ondansetron) include: (a) about 0.1-1000 μg/kg per application;(b) about 1 μg/kg; (c) about 2 μg/kg; (d) about 3 μg/kg; (e) about 4μg/kg; (f) about 5 μg/kg; (g) about 6 g/kg; (h) about 7 μg/kg; (i) about8 μg/kg; (j) about 9 μg/kg; (k) about 10, about 11, about 12, about 13,about 14, about 15, about 16, about 17, about 18, about 19, about 20,about 21, about 22, about 23, about 24, about 25, about 26, about 27,about 28, about 29, about 30, about 35, about 40, about 45, about 50,about 55, about 60, about 65, about 70, about 75, about 80, about 90, toabout 100 μg/kg; and, (1) about 100, about 200, about 300, about 400,about 500, about 600, about 700, about 800, about 900, to about 1000μg/kg.

Examples of the timing of administration include administering: (a) oncea day, (b) twice a day, (c) once a week, (d) twice a week, (e) once amonth, (f) twice a month, (g) once every 3 months, and (h) once every 6months.

In another aspect, the addictive disease or disorder is selected fromthe group consisting of alcohol-related diseases and disorders,obesity-related diseases and disorders, eating disorders, impulsecontrol disorders, nicotine-related disorders, amphetamine-relateddisorders, methamphetamine-related disorders, cannabis-relateddisorders, cocaine-related disorders, hallucinogen use disorders,inhalant-related disorders, benzodiazepine abuse or dependence relateddisorders, opioid-related disorders, gambling, sexual disorders,computer use related disorders, and electronic use related disorders.

In another aspect, the addictive disease or disorder is analcohol-related disease or disorder.

In another aspect, the alcohol-related disease or disorder is selectedfrom the group consisting of early onset alcoholism, late onsetalcoholism, alcohol-induced psychotic disorder with delusions, alcoholabuse, heavy drinking, excessive drinking, problem drinking, alcoholintoxication, alcohol withdrawal, alcohol intoxication delirium, alcoholwithdrawal delirium, alcohol-induced persisting dementia,alcohol-induced persisting amnestic disorder, alcohol dependence,alcohol-induced psychotic disorder with hallucinations, alcohol-inducedmood disorder, alcohol-induced or associated bipolar disorder,alcohol-induced or associated post traumatic stress disorder,alcohol-induced anxiety disorder, alcohol-induced sexual dysfunction,alcohol-induced sleep disorder, alcohol-induced or associated gamblingdisorder, alcohol-induced or associated sexual disorder, alcohol-relateddisorder not otherwise specified, alcohol intoxication, and alcoholwithdrawal.

In another aspect, the alcohol-related disease or disorder is earlyonset alcoholism. In another aspect, the alcohol-related disease ordisorder is late onset alcoholism.

In another aspect, the response from the treatment, comprises: areduction in drinking. Examples of reduction in drinking include, butare not limited to reduction of (a) heavy drinking, (b) excessivedrinking, (c) drinks/day, (d) percentage of subjects not drinkingheavily, (e) drinks/drinking day, (f) percentage of subjects with noheavy drinking, and (g) percentage of subjects who are abstinent.

In another aspect, the method reduces the quantity of alcohol consumedcompared with the amount of alcohol consumed before said treatment orcompared with a control subject not receiving said treatment. In anotheraspect, the alcohol consumption comprises heavy drinking or excessivedrinking.

In another aspect, the method improves the physical or psychologicalsequelae associated with alcohol consumption compared with a controlsubject not receiving said treatment.

In another aspect, the method increases the abstinence rate of saidsubject compared with a control subject not receiving said treatment.

In another aspect, the method reduces the average level of alcoholconsumption compared with the level before said treatment or comparedwith a control subject not receiving said treatment.

In another aspect, the method reduces alcohol consumption and increasesabstinence compared with the alcohol consumption and abstinence beforesaid treatment or compared with a control subject not receiving saidtreatment.

In another aspect, the subject is submitted to a psychosocial managementprogram.

In another aspect, the psychosocial management program is selected fromthe group consisting of Brief Behavioral Compliance EnhancementTreatment; Cognitive Behavioral Coping Skills Therapy; MotivationalEnhancement Therapy; Twelve-Step Facilitation Therapy; CombinedBehavioral Intervention; Medical Management; psychoanalysis;psychodynamic treatment; Biopsychosocial, Report, Empathy, Needs, DirectAdvice and Assessment; and, computer-delivered education or treatment.

In another aspect, the subject is further subjected to hypnosis oracupuncture.

In another aspect, effective amounts of at least two antagonists areadministered.

In another aspect, effective amounts of at least three antagonists areadministered.

In another aspect, the present invention provides a method of selectingpatients with an addictive disease or disorder who will be responsive totreatment with an antagonist of the serotonin receptor 5-HT₃,comprising: determining whether he patient's serotonin transporter geneSLC6A4 has:

-   -   (a) 3 or fewer of the genotypes selected from (Set (a)):        -   i. the LS or SS genotype of the functional polymorphism            serotonin transporter-linked polymorphic region 5-HTTLPR;        -   ii. the TG or GG genotype of the single nucleotide            polymorphism (SNP) rs1042173;        -   iii. the GG genotype of the SNP rs1176713;        -   iv. the AA genotype of the SNP rs1176719; and,        -   v. the GG genotype of the SNP rs1672717;    -   (b) 3 or more of the genotypes selected from (Set (b)):        -   v. the LL genotype of the functional polymorphism serotonin            transporter-linked polymorphic region 5-HTTLPR;        -   vi. the AG or GG genotype of SNP rs1176719;        -   vii. the GG or AG genotype of SNP rs1672717 (HTR3B); and,        -   viii. the AA genotype of SNP rs2276307 (HTR3B);    -   (c) 3 or more of the genotypes selected from (Set (c)):        -   i. the LL genotype of the functional polymorphism serotonin            transporter-linked polymorphic region 5-HTTLPR;        -   ii. the TT genotype of rs1042173 (SERT);        -   iii. the GT or GG genotype of rs10160548 (HTR3A);        -   iv. the GA or GG genotype of rs1176746 (HTR3B); and,        -   v. the GG genotype of rs12270070 (HTR3B).    -   (d) a genotype selected from (Set (d)):        -   i. the AG genotype of rs1150226;        -   ii. the AG genotype of rs1150226 and the AC genotype of            rs17614942;        -   iii. the AG genotype of rs1150226, the LL genotype of            5-HTTLPR, and the TT genotype of rs1042173;        -   iv. the AG genotype of rs1150226, the AC genotype of            rs17614942, the LL genotype of 5-HTTLPR, and the TT genotype            of rs1042173;        -   v. the AC genotype of rs17614942; or        -   vi. the AC genotype of rs17614942, the LL genotype of            5-HTTLPR, and the TT genotype of rs1042173;    -   (e) the AA genotype of rs1176719;    -   (f) the GG genotype of rs1176713;    -   (g) the AC genotype of rs17614942 and the LL genotype of        5-HTTLPR;    -   (h) the AG genotype of rs1150226 and at least one genotype        selected from:        -   iii. the AC genotype of rs17614942; and        -   iv. the LL genotype of 5-HTTLPR;    -   (i) the AA genotype of rs1176719 and at least one genotype        selected from:        -   iv. the AC genotype of rs17614942;        -   v. the LL genotype of 5-HTTLPR; and,        -   vi. the TT genotype of rs1042173;    -   (j) the AC genotype of rs17614942 and at least one genotype        selected from:        -   iv. the AA genotype of rs1176719;        -   v. the LL genotype of 5-HTTLPR; and,        -   vi. the TT genotype of rs1042173;    -   (k) the GG genotype of rs1176713 and at least one genotype        selected from:        -   iii. the LL genotype of 5-HTTLPR; and,        -   iv. the TT genotype of rs1042173;    -   (1) the GG genotype of rs1176713 and at least one genotype        selected from:        -   iii. the AC genotype of rs17614942; and,        -   iv. the LL genotype of 5-HTTLPR;    -   (m) the GG genotype of rs1176713 and at least one genotype        selected from:        -   iii. the AC genotype of rs17614942; and,        -   iv. the TT genotype of rs1042173;    -   (n) the GG genotype of rs1176713 and at least one genotype        selected from:        -   iii. the AG genotype of rs1150226; and,        -   iv. the TT genotype of rs1042173;    -   (o) the TT genotype of rs3758987 and at least one genotype        selected from genotype sets (i)-(iv);        -   v. the TT genotype of rs1042173;        -   vi. the AA genotype of rs2276307 and the LL genotype of            5-HTTLPR;        -   vii. the TT genotype of rs1042173 and the LL genotype of            5-HTTLPR; or        -   viii. the TT genotype of rs1042173, the AA genotype of            rs2276307, and the LL genotype of 5-HTTLPR; or    -   (p) the TC genotype of rs3758987 and at least one genotype        selected from genotype sets (i)-(iv);        -   v. the TT genotype of rs1042173;        -   vi. the AA genotype of rs2276307 and the LL genotype of            5-HTTLPR;        -   vii. the TT genotype of rs1042173 and the LL genotype of            5-HTTLPR; or        -   viii. the TT genotype of rs1042173, the AA genotype of            rs2276307, and the LL genotype of 5-HTTLPR.

In another aspect, the method of selecting, further comprises:

-   -   administering an antagonist of the serotonin receptor 5-HT₃ to        the patient, if the patient satisfies one of the (a)-(p)        criteria.

In another aspect, the present invention provides a method of treating apatient with an addictive disease or disorder, comprising:

-   -   a. determining whether the patient, in the patient's serotonin        transporter gene SLC6A4, satisfies one of the (a)-(p) criteria;        and,    -   b. administering an antagonist of the serotonin receptor 5-HT₃        to the patient, if the patient satisfies one of the (I)-(IV)        criteria.

In another aspect, the present invention provides a method of predictinga response to treatment for an addictive disease or disorder in asubject comprising: determining whether the patient, in the patient'sserotonin transporter gene SLC6A4, satisfies one of the (a)-(p)criteria.

In another aspect, the present invention further comprises:administering to a patient in need thereof a therapeutically effectiveamount of a second therapeutic agent (e.g., topiramate and/ornaltrexone). The present invention further encompasses the use ofadjunctive treatments and therapy such as psychosocial managementregimes, hypnosis, and acupuncture.

In another aspect, the present invention provides compositions andmethods for treating an addictive disease or disorder usingpharmaceutical compositions, comprising: effective amounts ofondansetron, topiramate and/or naltrexone.

In another aspect, the present invention provides a method of treatingan addictive disease or disorder, comprising: administering to a patientin need thereof a therapeutically effective amount of an antagonist ofthe serotonin receptor 5-HT₃, wherein the patient, in the patient'sserotonin transporter gene SLC6A4, is known to have the TG genotype ofthe single nucleotide polymorphism rs1042173. In another aspect, thepatient further is known to have the LL genotype of the functionalpolymorphism serotonin transporter-linked polymorphic region 5-HTTLPR ofthe serotonin transporter gene SLC6A4.

In another aspect, the present invention provides a method of predictinga response to treatment for an addictive disease or disorder in apatient comprising: determining whether the patient has the TG genotypeof the single nucleotide polymorphism rs1042173 of the serotonintransporter gene SLC6A4. The presence of the TG genotype is anindication that the patient will respond to treatment for an addictivedisease or disorder. In another aspect, the invention further comprises:determining if the patient further has the LL genotype of the functionalpolymorphism serotonin transporter-linked polymorphic region 5-HTTLPR ofthe serotonin transporter gene SLC6A4.

In another aspect, the method predicts the response to treatment with atleast one antagonist of the serotonin receptor 5-HT₃ (e.g.,ondansetron).

In another aspect, the present invention provides a method of selectingpatients with an addictive disease or disorder who will be responsive totreatment with an antagonist of the serotonin receptor 5-HT₃,comprising: determining whether the patient has the TG genotype of thesingle nucleotide polymorphism rs1042173 of the serotonin transportergene SLC6A4. In another aspect, the invention further comprises:determining if the patient further has the LL genotype of the functionalpolymorphism serotonin transporter-linked polymorphic region 5-HTTLPR ofthe serotonin transporter gene SLC6A4.

In another aspect, the present invention provides a method of treating apatient with an addictive disease or disorder, comprising:

-   -   a) determining whether the patient has the TG genotype of the        single nucleotide polymorphism rs1042173 of the serotonin        transporter gene SLC6A4; and    -   b) administering at least one antagonist of the serotonin        receptor 5-HT₃ to the patient having the TG genotype.

In another aspect, the invention further comprises: determining if thepatient further has the LL genotype of the functional polymorphismserotonin transporter-linked polymorphic region 5-HTTLPR of theserotonin transporter gene SLC6A4.

In another aspect, the present invention provides a method of treatingan addictive disease or disorder, comprising: administering to a patientin need thereof a therapeutically effective amount of an antagonist ofthe serotonin receptor 5-HT₃, wherein the patient, in the patient'sserotonin transporter gene SLC6A4, is known to have the AA or ACgenotype of the single nucleotide polymorphism rs17614942 (5HT3b). Inanother aspect, the patient further is known to have the LL genotype ofthe functional polymorphism serotonin transporter-linked polymorphicregion 5-HTTLPR of the serotonin transporter gene SLC6A4.

In another aspect, the present invention provides a method of predictinga response to treatment for an addictive disease or disorder in apatient comprising: determining whether the patient has the AA or ACgenotype of the single nucleotide polymorphism rs17614942 (5HT3b) of theserotonin transporter gene SLC6A4. In another aspect, the inventionfurther comprises: determining if the patient further has the LLgenotype of the functional polymorphism serotonin transporter-linkedpolymorphic region 5-HTTLPR of the serotonin transporter gene SLC6A4.

In another aspect, the method predicts the response to treatment with atleast one antagonist of the serotonin receptor 5-HT₃ (e.g.,ondansetron).

In another aspect, the present invention provides a method of selectingpatients with an addictive disease or disorder who will be responsive totreatment with an antagonist of the serotonin receptor 5-HT₃,comprising: determining whether the patient has the AA or AC genotype ofthe single nucleotide polymorphism rs17614942 (5HT3b) of the serotonintransporter gene SLC6A4. In another aspect, the invention furthercomprises: determining if the patient further has the LL genotype of thefunctional polymorphism serotonin transporter-linked polymorphic region5-HTTLPR of the serotonin transporter gene SLC6A4.

In another aspect, the present invention provides a method of treating apatient with an addictive disease or disorder, comprising:

-   -   a) determining whether the patient has the AA or AC genotype of        the single nucleotide polymorphism rs17614942 (5HT3b) of the        serotonin transporter gene SLC6A4; and    -   b) administering at least one antagonist of the serotonin        receptor 5-HT₃ to the patient having the TG genotype.

In another aspect, the invention further comprises: determining if thepatient further has the LL genotype of the functional polymorphismserotonin transporter-linked polymorphic region 5-HTTLPR of theserotonin transporter gene SLC6A4.

In another aspect, the present invention provides a method of treatingan addictive disease or disorder, comprising: administering to a patientin need thereof a therapeutically effective amount of an antagonist ofthe serotonin receptor 5-HT₃, wherein the patient, in the patient'sserotonin transporter gene SLC6A4, is known to have the AA or ACgenotype of the single nucleotide polymorphism rs4938056 (5HT3b). Inanother aspect, the patient further is known to have the LL genotype ofthe functional polymorphism serotonin transporter-linked polymorphicregion 5-HTTLPR of the serotonin transporter gene SLC6A4.

In another aspect, the present invention provides a method of predictinga response to treatment for an addictive disease or disorder in apatient comprising: determining whether the patient has the AA or ACgenotype of the single nucleotide polymorphism rs4938056 (5HT3b) of theserotonin transporter gene SLC6A4. In another aspect, the inventionfurther comprises: determining if the patient further has the LLgenotype of the functional polymorphism serotonin transporter-linkedpolymorphic region 5-HTTLPR of the serotonin transporter gene SLC6A4.

In another aspect, the method predicts the response to treatment with atleast one antagonist of the serotonin receptor 5-HT₃ (e.g.,ondansetron).

In another aspect, the present invention provides a method of selectingpatients with an addictive disease or disorder who will be responsive totreatment with an antagonist of the serotonin receptor 5-HT₃,comprising: determining whether the patient has the AA or AC genotype ofthe single nucleotide polymorphism rs4938056 (5HT3b) of the serotonintransporter gene SLC6A4. In another aspect, the invention futhercomprises: determining if the patient further has the LL genotype of thefunctional polymorphism serotonin transporter-linked polymorphic region5-HTTLPR of the serotonin transporter gene SLC6A4.

In another aspect, the present invention provides a method of treating apatient with an addictive disease or disorder, comprising:

-   -   a) determining whether the patient has the AA or AC genotype of        the single nucleotide polymorphism rs4938056 (5HT3b) of the        serotonin transporter gene SLC6A4; and    -   b) administering at least one antagonist of the serotonin        receptor 5-HT₃ to the patient having the TG genotype.

In another aspect, the invention further comprises: determining if thepatient further has the LL genotype of the functional polymorphismserotonin transporter-linked polymorphic region 5-HTTLPR of theserotonin transporter gene SLC6A4.

One of ordinary skill in the art will appreciate that in some instancesa patient being treated for an addictive disorder is not necessarilydependent. Such patients include, for example, patients who abusealcohol, drink heavily, drink excessively, are problem drinkers, or areheavy drug users. The present invention provides compositions andmethods for treating or preventing these behaviors in non-dependentpatients.

In another aspect, the present invention provides compositions andmethods for improving the physical or psychological sequelae associatedwith alcohol consumption compared with a control subject not receivingthe treatment.

In another aspect, the present invention provides compositions andmethods for increasing the abstinence rate of a subject compared with acontrol subject not receiving the treatment.

In another aspect, the present invention provides compositions andmethods for reducing the average level of alcohol consumption in asubject compared with the level of alcohol consumption before thetreatment or compared with the level of alcohol consumption by a controlsubject not receiving the treatment.

In another aspect, the present invention provides compositions andmethods for reducing alcohol consumption and for increasing abstinencecompared with the alcohol consumption by the subject before treatment orwith a control subject not receiving the treatment.

In another aspect, the present invention provides compositions andmethods for treating a subject with a predisposition to early-onsetalcoholism.

In another aspect, the present invention provides compositions andmethods for treating a subject with a predisposition to late-onsetalcoholism.

One of ordinary skill in the art will appreciate that there are multipleparameters or characteristics of alcohol consumption which maycharacterize a subject afflicted with an alcohol-related disease ordisorder. It will also be appreciated that combination therapies may beeffective in treating more than one parameter, and that there aremultiple ways to analyze the effectiveness of treatment. The parametersanalyzed when measuring alcohol consumption or frequency of alcoholconsumption include, but are not limited to, heavy drinking days, numberof heavy drinking days, average drinking days, number of drinks per day,days of abstinence, number of individuals not drinking heavily orabstinent over a given time period, and craving. Both subjective andobjective measures can be used to analyze the effectiveness oftreatment. For example, a subject can self-report according toguidelines and procedures established for such reporting. The procedurescan be performed at various times before, during, and after treatment.Additionally, assays are available for measuring alcohol consumption.These assays include breath alcohol meter readings, measuring serum CDTand GGT levels, and measuring 5-HTOL urine levels.

When combination therapy is used, the timing of administration of thecombination can vary. First example, the first compound and a secondcompound can be administered nearly simultaneously. Other examplesinclude (a) the first compound being administered prior to the secondcompound, (b) the first compound being administered subsequent to thesecond compound, and (c) if three or more compounds are administered,one of ordinary skill in the art will appreciate that the three or morecompounds can be administered simultaneously or in varying order.

In another aspect, the present invention provides a method of treating,comprising administering at least two compounds selected from the groupconsisting of topiramate, ondansetron, and naltrexone. In one aspect,topiramate and ondansetron are used.

Because the serotonin system has intimate connections and is modulatedin the brain by other neurotransmitters, particularly dopamine, GABA,glutamate, opioids, and cannabinoid, the present invention alsoencompasses the use of medications and drugs that affect the structureand function of these other neurotransmitters when combined with anyserotonergic agent (including ondansetron). In one aspect, thecombination is efficacious for individuals with the polymorphismsdescribed herein. In another aspect, the present invention providescompositions, compounds and methods that are associated with theseco-modulating neurotransmitters (i.e., dopamine, GABA, glutamate,opioids, and cannabinoid), including, but not limited to, topiramate,baclofen, gabapentin, naltrexone, nalmefene, and rimonabant-incombination with any serotonergic agent (including but not limited toondansetron, selective serotonin re-uptake blockers, and other agonistsor antagonists of other serotonin receptors or moieties) can produce atherapeutic effect to improve the clinical outcomes for individuals whouse, abuse, misuse, or are dependent on alcohol. Because abused drugsare predicted to work through similar mechanisms, the present inventionfurther provides combinations of these co-modulating drugs with anyother serotonergic agent to be used to treat individuals with anysubstance use, abuse, misuse, dependence, or habit-forming behavior withthe polymorphisms described herein or anywhere else in the serotonergicor co-modulating neurotransmitter systems (i.e., dopamine, GABA,glutamate, opioids, and cannabinoid), either alone or in combination.

In a further aspect, the combination pharmacotherapy treatment is usedin conjunction with behavioral modification or therapy.

The dosage of the active compound(s) being administered will depend onthe condition being treated, the particular compound, and other clinicalfactors such as age, sex, weight, and health of the subject beingtreated, the route of administration of the compound(s), and the type ofcomposition being administered (tablet, gel cap, capsule, solution,suspension, inhaler, aerosol, elixir, lozenge, injection, patch,ointment, cream, etc.). It is to be understood that the presentinvention has application for both human and veterinary use.

The drugs can be administered in formulations that contain all drugsbeing used, or the drugs can be administered separately. In some cases,it is anticipated that multiple doses/times of administration will beuseful. The present invention further provides for varying the length oftime of treatment.

In another aspect, the present invention provides a compositioncomprising: an antagonist of the serotonin receptor 5-HT₃. In anotheraspect, the composition further comprises a second therapeutic agent. Inanother aspect, the composition further comprises a third therapeuticagent.

Topiramate (C₁₂H₂₁NO₈S; IUPAC name:2,3:4,5-Bis-O-(1-methylethylidene)-beta-D-fructopyranose sulfamate; CASRegistry No. 97240-79-4) is disclosed herein as a drug useful incombination drug therapy. Examples of topiramate dosages include: (a)about 15, about 25, about 35, about 35, about 55, about 65, about 75,about 85, about 95, about 100, about 200, about 300, about 400, about500, about 600, about 700, about 800, about 900, about 1000, about 1100,about 1200, about 1300, about 1400, about 1500, about 1600, about 1700,about 1800, about 1900, about 2000, about 2100, about 2200, about 2300,about 2400, to about 2500 mg/day, (b) about 25-1000 mg/day, (c) about50, about 60, about 70, about 80, about 90, about 100, about 200, about300, about 400, to about 500 mg/day, (f) about 275 mg/day, (g) about 1mg/day, (h) about 1 mg/kg, (i) about 10 mg/kg, (j) about 100 mg/kg, and(k) about 0.1, about 0.2, about 0.3., about 0.4., about 0.5, about 0.6,about 0.7, about 0.8, about 0.9, about 1.0, about 2, about 3, about 4,about 5, about 6, about 7, about 8, about 9, about 10, about 20, about30, about 40, about 50, about 60, about 70, about 80, about 90 to about100 mg/kg/day.

An aspect of psychotropic drugs is to produce weight gain. Theseincreases in weight gain can induce a range of metabolic problemsincluding abnormal sugar, fat, and carbohydrate metabolism. Becausetopiramate can cause weight loss and improve endocrine function, it isproposed herein that topiramate may be used to ameliorate weight gaincaused by other psychotropic drugs with which it is combined as well asalcohol and any other abused drugs.

An adverse event of topiramate is cognitive impairment. In the generalpopulation, this is reported by 2.4% of individuals who take topiramate(Johnson & Johnson Pharmaceutical Research & Development. Investigator'sBrochure: Topiramate (RWJ-17021-000), 10th ed.; December 2005). In thesubstance abuse field, the occurrence rate of cognitive impairment isabout 18.7% (Johnson B A, Ait-Daoud N, Bowden C L et al. Oral topiramatefor treatment of alcohol dependence: a randomized controlled trial.Lancet 2003, 361:1677-1685). Topiramate-associated cognitive effects aredue to its anti-glutaminergic properties. It is, therefore, not obviousthat ondansetron, a serotonin-3 receptor antagonist, will alleviatethese complaints of cognitive impairment. Ondansetron appears to havecholinergic effects, perhaps through interactions with the GABA systemthat seem to ameliorate topiramate-associated cognitive impairment.Hence, the rate of cognitive impairment reported by this triplecombination would be less than that for topiramate on its own.

Ondansetron (C₁₈H₁₉N₃O; CAS Registry No. 99614-02-5; IUPAC name:9-methyl-3-[(2-methyl-1H-imidazol-1-yl)methyl]-1,2,3,9-tetrahydrocarbazol-4-one)is disclosed herein as a drug useful alone or as part of combinationdrug therapy. Ondansetron is a 5-HT₃ receptor antagonist and hasfunctionally opposite effects to SSRIs and blocks serotonin agonism atthe 5-HT₃ receptor. The dosage and treatment regimen for administeringondansetron when it is being used as one compound of a combinationtherapy can be varied based on the other drug or drugs with which it isbeing administered, or based on other criteria such as the age, sex,health, and weight of the subject.

The present invention further provides for the use of other drugs suchas naltrexone (C₂₀H₂₃NO₄;17-(Cyclopropylmethyl)-4,5a-epoxy-3,14-dihydroxymorphinan-6-onehydrochloride; CAS Registry No. 16590-41-3) as part of the drugcombination therapy disclosed herein. Examples of naltrexone dosagesinclude: (a) 10 mg/day, (b) 50 mg/day, (c) 100 mg/day, (d) 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250,to 300 mg per application, (e) 10-50 mg per application, and (f) 25 mgper application.

Naltrexone also has adverse events—nausea and vomiting—that reducecompliance to it. Indeed, about 15% of individuals in alcohol trials areunable to tolerate a naltrexone dose of 50 mg/day. This has led to thedevelopment of depot formulations that release naltrexone slowly toreduce the incidence of nausea and vomiting. Nevertheless, these depotformulation(s) appear to have similar compliance rates to the oral formof the medication. Ondansetron reduces nausea and decreases vomiting byslowing gut motility. Therefore, a combination that adds ondansetron tonaltrexone will diminish the nausea and vomiting caused by naltrexone.This is a therapeutic advance because many more people will be able totolerate the treatment due to increased compliance, and higher dosesthan the typically administered naltrexone dose of 50 mg/day can begiven to improve the therapeutic response.

The present invention provides for multiple methods for delivering thecompounds of the invention. The compounds may be provided, for example,as pharmaceutical compositions in multiple formats as well, including,but not limited to, tablets, capsules, pills, lozenges, syrups,ointments, creams, elixirs, suppositories, suspensions, inhalants,injections (including depot preparations), and liquids.

The invention further encompasses treating and preventing obesity, i.e.,for affecting weight loss and preventing weight gain. Obesity is adisorder characterized by the accumulation of excess fat in the body.Obesity has been recognized as one of the leading causes of disease andis emerging as a global problem. Increased instances of complicationssuch as hypertension, non-insulin-dependent diabetes mellitus,arteriosclerosis, dyslipidemia, certain forms of cancer, sleep apnea,and osteoarthritis have been related to increased instances of obesityin the general population In one aspect, the invention encompassesadministering to a subject in need thereof a combination therapy toinduce weight loss. For example, subjects having a BMI of greater thanabout 25 (25.0-29.9 is considered overweight) are identified fortreatment. In one aspect, the individuals have a BMI of greater than 30(30 and above is considered obese). In another aspect, a subject may betargeted for treatment to prevent weight gain. In one embodiment, anindividual is instructed to take at least one compound of the inventionat least once daily and at least a second compound of the invention atleast once daily. The compound may be in the form of, for example, atablet, a lozenge, a liquid, etc. In one aspect, a third compound isalso taken daily. In one embodiment, compounds may be taken more thanonce daily. In another embodiment, compounds are taken less than oncedaily. The dosages can be determined based on what is known in the artor what is determined to be best for a subject of that age, sex, health,weight, etc. Compounds useful for treating obesity according to themethods of the invention, include, but are not limited to, topiramate,naltrexone, and ondansetron. See Weber (U.S. Pat. Pub. No. 20070275970)and McElroy (U.S. Pat. No. 6,323,236) for additional information andtechniques for administering drugs useful for treating obesity,addictive disorders, and impulse control disorders, and for determiningdosage schemes.

Pharmaceutically-acceptable base addition salts can be prepared frominorganic and organic bases. Salts derived from inorganic bases, includeby way of example only, sodium, potassium, lithium, ammonium, calciumand magnesium salts. Salts derived from organic bases include, but arenot limited to, salts of primary, secondary and tertiary amines, such asalkyl amines, dialkyl amines, trialkyl amines, substituted alkyl amines,di(substituted alkyl) amines, tri(substituted alkyl) amines, alkenylamines, dialkenyl amines, trialkenyl amines, substituted alkenyl amines,di(substituted alkenyl) amines, tri(substituted alkenyl) amines,cycloalkyl amines, di(cycloalkyl) amines, tri(cycloalkyl) amines,substituted cycloalkyl amines, disubstituted cycloalkyl amines,trisubstituted cycloalkyl amines, cycloalkenyl amines, di(cycloalkenyl)amines, tri(cycloalkenyl) amines, substituted cycloalkenyl amines,disubstituted cycloalkenyl amines, trisubstituted cycloalkenyl amines,aryl amines, diaryl amines, triaryl amines, heteroaryl amines,diheteroaryl amines, triheteroaryl amines, heterocyclic amines,diheterocyclic amines, triheterocyclic amines, mixed di- and tri-amineswhere at least two of the substituents on the amine are different andare selected from the group consisting of alkyl, substituted alkyl,alkenyl, substituted alkenyl, cycloalkyl, substituted cycloalkyl,cycloalkenyl, substituted cycloalkenyl, aryl, heteroaryl, heterocyclic,and the like. Also included are amines where the two or threesubstituents, together with the amino nitrogen, form a heterocyclic orheteroaryl group. Examples of suitable amines include, by way of exampleonly, isopropylamine, trimethyl amine, diethyl amine, tri(iso-propyl)amine, tri(n-propyl) amine, ethanolamine, 2-dimethylaminoethanol,tromethamine, lysine, arginine, histidine, caffeine, procaine,hydrabamine, choline, betaine, ethylenediamine, glucosamine,N-alkylglucamines, theobromine, purines, piperazine, piperidine,morpholine, N-ethylpiperidine, and the like. It should also beunderstood that other carboxylic acid derivatives would be useful in thepractice of this invention, for example, carboxylic acid amides,including carboxamides, lower alkyl carboxamides, dialkyl carboxamides,and the like.

Pharmaceutically acceptable acid addition salts may be prepared frominorganic and organic acids. Salts derived from inorganic acids includehydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid,phosphoric acid, and the like. Salts derived from organic acids includeacetic acid, propionic acid, glycolic acid, pyruvic acid, oxalic acid,malic acid, malonic acid, succinic acid, maleic acid, fumaric acid,tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid,methanesulfonic acid, ethanesulfonic acid, p-toluene-sulfonic acid,salicylic acid, and the like.

Psychosocial Intervention and Management

The drug combination treatments of the present invention can be furthersupplemented by providing to subjects a form of psychosocialintervention and/or management, such as Brief Behavioral ComplianceEnhancement Treatment (BBCET). BBCET, a standardized, manual-guided,brief (i.e., delivered in about 15 minutes), psychosocial adherenceenhancement procedure, emphasizes that medication compliance is crucialto changing participants' drinking behavior (Johnson et al., BriefBehavioral Compliance Enhancement Treatment (BBCET) manual. In: JohnsonB A, Ruiz P, Galanter M, eds. Handbook of clinical alcoholism treatment.Baltimore, Md.: Lippincott Williams & Wilkins; 2003, 282-301). Briefinterventions (Edwards et al., J. Stud. Alcohol. 1977, 38:1004-1031)such as BBCET, have been shown to benefit treatment of alcoholdependence. BBCET was modeled on the clinical management condition inthe National Institute of Mental Health collaborative depression trial,which was used as an adjunct to the medication condition for that study(Fawcett et al. Psychopharmacol Bull. 1987, 23:309-324). BBCET has beenused successfully as the psychosocial treatment platform in thesingle-site and multi-site efficacy trials of topiramate for treatingalcohol dependence (Johnson et al., Lancet. 2003, 361:1677-1685; Johnsonet al., JAMA, 2007, 298:1641-1651). It is delivered by trainedclinicians, including nurse practitioners and other non-specialists.Uniformity and consistency of BBCET delivery are ensured by ongoingtraining and supervision. BBCET is copyrighted material (Johnson et al.,Brief Behavioral Compliance Enhancement Treatment (BBCET) manual. In:Johnson B A, Ruiz P, Galanter M, eds. Handbook of clinical alcoholismtreatment. Baltimore, Md.: Lippincott Williams & Wilkins; 2003,282-301).

The present invention further encompasses the use of psychosocialmanagement regimens other than BBCET, including, but not limited to,Cognitive Behavioral Coping Skills Therapy (CBT) (Project MATCH ResearchGroup. Matching Alcoholism Treatments to Client Heterogeneity: ProjectMATCH posttreatment drinking outcomes. J Stud Alcohol. 1997; 58:7-29),Motivational Enhancement Therapy (MET) (Project MATCH Research Group.Matching Alcoholism Treatments to Client Heterogeneity: Project MATCHposttreatment drinking outcomes. J. Stud. Alcohol. 1997, 58:7-29),Twelve-Step Facilitation Therapy (TSF) (Project MATCH Research Group.Matching Alcoholism Treatments to Client Heterogeneity: Project MATCHposttreatment drinking outcomes. J. Stud. Alcohol. 1997, 58:7-29),Combined Behavioral Intervention (CBI), (Anton et al., JAMA, 2006,295:2003-2017) Medical Management (MM) (Anton et al., JAMA, 2006,295:2003-2017), or the Biopsychosocial, Report, Empathy, Needs, Directadvice, and Assessment (BRENDA) model (Garbutt et al., JAMA, 2005,293:1617-1625). The present invention further encompasses the use ofalternative interventions such as hypnosis or acupuncture to assist intreating an addictive disease or disorder.

The psychosocial management programs can be used before, during, andafter treating the subject with the combination drug therapy of theinvention.

One of ordinary skill in the art will recognize that psychosocialmanagement procedures, as well as alternative interventions such ashypnosis or acupuncture, can also be used in conjunction withcombination drug therapy to treat addictive and impulse-relateddisorders other than alcohol-related diseases and disorders.

The present invention further encompasses the use of combinationpharmacotherapy and behavioral (psychosocial) intervention or trainingto treat other addictive and/or impulse control disorders.

For example, binge eating disorder (BED) is characterized by discreteperiods of binge eating during which large amounts of food are consumedin a discrete period of time and a sense of control over eating isabsent. Persons with bulimia nervosa have been reported to haveelectroencephalographic abnormalities and to display reduced bingeeating in response to the anti-epileptic drug phenytoin. In addition, incontrolled trials in patients with epilepsy, topiramate was associatedwith suppression of appetite and weight loss unrelated to binge eating.Ondansetron has been shown to reduce binge eating.

BED is a subset of a larger classification of mental disorders broadlydefined as Impulse Control Disorders (ICDs) characterized by harmfulbehaviors performed in response to irresistible impulses. It has beensuggested that ICDs may be related to obsessive-compulsive disorder orsimilarly, maybe forms of obsessive-compulsive disorders. It has alsobeen hypothesized that ICDs may be related to mood disorder or may beforms of affective spectrum disorder, a hypothesized family of disorderssharing at least one common physiologic abnormality with majordepression. In the Diagnostic and Statistical Manual of Mental Disorders(DSM-IV), the essential feature of an ICD is the failure to resist animpulse, drive, or temptation to perform an act that is harmful to theperson or to others. For most ICDs, the individual feels an increasingsense of tension or arousal before committing the act, and thenexperiences pleasure, gratification, or release at the time ofcommitting the act. After the act is performed, there may or may not beregret or guilt. ICDs are listed in a residual category, the ICDs NotElsewhere Classified, which includes intermittent explosive disorder(IED), kleptomania, pathological gambling, pyromania, trichotillomania,and ICDs not otherwise specified (NOS). Examples of ICDs NOS arecompulsive buying or shopping, repetitive self-mutilation, nonparaphilicsexual addictions, severe nail biting, compulsive skin picking,personality disorders with impulsive features, attentiondeficit/hyperactivity disorder, eating disorders characterized by bingeeating, and substance use disorders.

Many drugs can cause physical and/or psychological addiction. Those mostwell known drugs include opiates, such as heroin, opium and morphine;sympathomimetics, including cocaine and amphetamines;sedative-hypnotics, including alcohol, benzodiazepines, andbarbiturates; and nicotine, which has effects similar to opioids andsympathomimetics. Drug addiction is characterized by a craving orcompulsion for taking the drug and an inability to limit its intake.Additionally, drug dependence is associated with drug tolerance, theloss of effect of the drug following repeated administration, andwithdrawal, the appearance of physical and behavioral symptoms when thedrug is not consumed. Sensitization occurs if repeated administration ofa drug leads to an increased response to each dose. Tolerance,sensitization, and withdrawal are phenomena evidencing a change in thecentral nervous system resulting from continued use of the drug. Thischange motivates the addicted individual to continue consuming the drugdespite serious social, legal, physical, and/or professionalconsequences.

Attention-deficit disorders include, but are not limited to,Attention-Deficit/Hyperactivity Disorder, Predominately InattentiveType; Attention-Deficit/Hyperactivity Disorder, PredominatelyHyperactivity-Impulsive Type; Attention-Deficit/Hyperactivity Disorder,Combined Type; Attention-Deficit/Hyperactivity Disorder not otherwisespecified (NOS); Conduct Disorder; Oppositional Defiant Disorder; andDisruptive Behavior Disorder not otherwise specified (NOS).

Depressive disorders include, but are not limited to, Major DepressiveDisorder, Recurrent; Dysthymic Disorder; Depressive Disorder nototherwise specified (NOS); and Major Depressive Disorder, SingleEpisode.

Parkinson's disease includes, but is not limited to, neuroleptic-inducedparkinsonism.

Addictive disorders include, but are not limited to, eating disorders,impulse control disorders, alcohol-related disorders, nicotine-relateddisorders, amphetamine-related disorders, cannabis-related disorders,cocaine-related disorders, gambling, sexual disorders, hallucinogen usedisorders, inhalant-related disorders, and opioid-related disorders, allof which arc further subclassified as listed below.

Eating disorders include, but are not limited to, Bulimia Nervosa,Nonpurging Type; Bulimia Nervosa, Purging Type; and Eating Disorder nototherwise specified (NOS).

Impulse control disorders include, but are not limited to, IntermittentExplosive Disorder, Kleptomania, Pyromania, Pathological Gambling,Trichotillomania, and Impulse Control Disorder not otherwise specified(NOS).

Nicotine-related disorders include, but are not limited to, NicotineDependence, Nicotine Withdrawal, and Nicotine-Related Disorder nototherwise specified (NOS).

Amphetamine-related disorders include, but are not limited to,Amphetamine Dependence, Amphetamine Abuse, Amphetamine Intoxication,Amphetamine Withdrawal, Amphetamine Intoxication Delirium,Amphetamine-Induced Psychotic Disorder with delusions,Amphetamine-Induced Psychotic Disorders with hallucinations,Amphetamine-Induced Mood Disorder, Amphetamine-Induced Anxiety Disorder,Amphetamine-Induced Sexual Dysfunction, Amphetamine-Induced SleepDisorder, Amphetamine Related Disorder not otherwise specified (NOS),Amphetamine Intoxication, and Amphetamine Withdrawal.

Cannabis-related disorders include, but are not limited to, CannabisDependence; Cannabis Abuse; Cannabis Intoxication; Cannabis IntoxicationDelirium; Cannabis-Induced Psychotic Disorder, with delusions;Cannabis-Induced Psychotic Disorder with hallucinations;Cannabis-Induced Anxiety Disorder; Cannabis-Related Disorder nototherwise specified (NOS); and Cannabis Intoxication.

Cocaine-related disorders include, but are not limited to, CocaineDependence, Cocaine Abuse, Cocaine Intoxication, Cocaine Withdrawal,Cocaine Intoxication Delirium, Cocaine-Induced Psychotic Disorder withdelusions, Cocaine-Induced Psychotic Disorders with hallucinations,Cocaine-Induced Mood Disorder, Cocaine-Induced Anxiety Disorder,Cocaine-Induced Sexual Dysfunction, Cocaine-Induced Sleep Disorder,Cocaine-Related Disorder not otherwise specified (NOS), CocaineIntoxication, and Cocaine Withdrawal.

Hallucinogen-use disorders include, but are not limited to, HallucinogenDependence, Hallucinogen Abuse, Hallucinogen Intoxication, HallucinogenWithdrawal, Hallucinogen Intoxication Delirium, Hallucinogen-InducedPsychotic Disorder with delusions, Hallucinogen-Induced PsychoticDisorder with hallucinations, Hallucinogen-Induced Mood Disorder,Hallucinogen-Induced Anxiety Disorder, Hallucinogen-Induced SexualDysfunction, Hallucinogen-Induced Sleep Disorder, Hallucinogen RelatedDisorder not otherwise specified (NOS), Hallucinogen Intoxication, andHallucinogen Persisting Perception Disorder (Flashbacks).

Inhalant-related disorders include, but are not limited to, InhalantDependence; Inhalant Abuse; Inhalant Intoxication; Inhalant IntoxicationDelirium; Inhalant-Induced Psychotic Disorder, with delusions;Inhalant-Induced Psychotic Disorder with hallucinations;Inhalant-Induced Anxiety Disorder; Inhalant-Related Disorder nototherwise specified (NOS); and Inhalant Intoxication.

Opioid-related disorders include, but are not limited to, OpioidDependence, Opioid Abuse, Opioid Intoxication, Opioid IntoxicationDelirium, Opioid-Induced Psychotic Disorder, with delusions,Opioid-Induced Psychotic Disorder with hallucinations, Opioid-InducedAnxiety Disorder, Opioid-Related Disorder not otherwise specified (NOS),Opioid Intoxication, and Opioid Withdrawal.

Tic disorders include, but are not limited to, Tourette's Disorder,Chronic Motor or Vocal Tic Disorder, Transient Tic Disorder, TicDisorder not otherwise specified (NOS), Stuttering, Autistic Disorder,and Somatization Disorder.

The present invention further encompasses the treatment of at least twoaddictive diseases or disorders or impulse control disorderssimultaneously. For example, the present invention provides for thesimultaneous treatment of alcohol related disorders and weight control(see Examples).

The present invention also encompasses the use of the compounds andcombination therapies of the invention in circumstances where mandatorytreatment may be applicable. For example, a court may require that asubject be treated or take part in a treatment program using compoundsor combination therapies of the invention as part of a mandated therapyrelated to alcohol abuse, excessive drinking, drug use, etc. Moreparticularly, the invention encompasses forensic uses where a courtwould require a subject who has been convicted of driving under theinfluence to be subjected to the methods of the invention as part of acondition of bail, probation, treatment, etc.

The invention also encompasses the use of pharmaceutical compositionscomprising compounds of the invention to practice the methods of theinvention, the compositions comprising at least one appropriate compoundand a pharmaceutically-acceptable carrier.

Other methods useful for the practice of the invention can be found, forexample, in U.S. Pat. Pub. No. 2006/0173064 (Lippa et al.), U.S. Pat.No. 6,323,236 (McElroy), U.S. Pat. Pub. No. 2007/0275970, PCTapplication PCT/US/2008/052628 (Johnson et al.) filed Jan. 31, 2008, andPCT application PCT/US/2007/088100 (Johnson and Tiouririne), filed Dec.19, 2007.

The pharmaceutical compositions useful for practicing the invention maybe, for example, administered to deliver a dose of between 1 ng/kg/dayand 100 mg/kg/day.

Pharmaceutical compositions that are useful in the methods of theinvention may be administered, for example, systemically in oral solidformulations, or as ophthalmic, suppository, aerosol, topical or othersimilar formulations. In addition to the appropriate compounds, suchpharmaceutical compositions may contain pharmaceutically-acceptablecarriers and other ingredients known to enhance and facilitate drugadministration. Other possible formulations, such as nanoparticles,liposomes, resealed erythrocytes, and immunologically based systems mayalso be used to administer an appropriate compound, or an analog,modification, or derivative thereof according to the methods of theinvention.

Compounds which are identified using any of the methods described hereinmay be formulated and administered to a subject for treatment of thediseases disclosed herein. One of ordinary skill in the art willrecognize that these methods will be useful for other diseases,disorders, and conditions as well.

The invention encompasses the preparation and use of pharmaceuticalcompositions comprising a compound useful for treatment of the diseasesdisclosed herein as an active ingredient. Such a pharmaceuticalcomposition may consist of the active ingredient alone, in a formsuitable for administration to a subject, or the pharmaceuticalcomposition may comprise the active ingredient and one or morepharmaceutically acceptable carriers, one or more additionalingredients, or some combination of these. The active ingredient may bepresent in the pharmaceutical composition in the form of aphysiologically acceptable ester or salt, such as in combination with aphysiologically acceptable cation or anion, as is well known in the art.

The formulations of the pharmaceutical compositions described herein maybe prepared by any method known or hereafter developed in the art ofpharmacology. In general, such preparatory methods include the step ofbringing the active ingredient into association with a carrier or one ormore other accessory ingredients, and then, if desirable, shaping orpackaging the product into a desired single- or multi-dose unit.

Although the descriptions of pharmaceutical compositions provided hereinare principally directed to pharmaceutical compositions which aresuitable for ethical administration to humans, it will be understood bythe skilled artisan that such compositions are generally suitable foradministration to animals of all sorts. Modification of pharmaceuticalcompositions suitable for administration to humans in order to renderthe compositions suitable for administration to various animals is wellunderstood, and the ordinarily skilled veterinary pharmacologist candesign and perform such modification with merely ordinary, if any,experimentation. Subjects to which administration of the pharmaceuticalcompositions of the invention is contemplated include, but are notlimited to, humans and other primates, mammals including commerciallyrelevant mammals such as cattle, pigs, horses, sheep, cats, and dogs,and birds including commercially relevant birds such as chickens, ducks,geese, and turkeys.

One type of administration encompassed by the methods of the inventionis parenteral administration, which includes, but is not limited to,administration of a pharmaceutical composition by injection of thecomposition, by application of the composition through a surgicalincision, by application of the composition through a tissue-penetratingnon-surgical wound, and the like. In particular, parenteraladministration is contemplated to include, but is not limited to,subcutaneous, intraperitoneal, intramuscular, and intrasternalinjection, and kidney dialytic infusion techniques

Pharmaceutical compositions that are useful in the methods of theinvention may be prepared, packaged, or sold in formulations suitablefor oral, rectal, vaginal, parenteral, topical, pulmonary, intranasal,inhalation, buccal, ophthalmic, intrathecal or another route ofadministration. Other contemplated formulations include projectednanoparticles, liposomal preparations, resealed erythrocytes containingthe active ingredient, and immunologically-based formulations.

A pharmaceutical composition of the invention may be prepared, packaged,or sold in bulk, as a single unit dose, or as a plurality of single unitdoses. As used herein, a “unit dose” is a discrete amount of thepharmaceutical composition comprising a predetermined amount of theactive ingredient. The amount of the active ingredient is generallyequal to the dosage of the active ingredient which would be administeredto a subject, or a convenient fraction of such a dosage such as, forexample, one-half or one-third of such a dosage.

The relative amounts of the active ingredient, the pharmaceuticallyacceptable carrier, and any additional ingredients in a pharmaceuticalcomposition of the invention will vary, depending upon the identity,size, and condition of the subject treated and further depending uponthe route by which the composition is to be administered. By way ofexample, the composition may comprise between 0.1% and 100% (w/w) activeingredient.

In addition to the active ingredient, a pharmaceutical composition ofthe invention may further comprise one or more additionalpharmaceutically active agents. Particularly contemplated additionalagents include anti-emetics and scavengers such as cyanide and cyanatescavengers.

Controlled- or sustained-release formulations of a pharmaceuticalcomposition of the invention may be made using conventional technology.

A formulation of a pharmaceutical composition of the invention suitablefor oral administration may be prepared, packaged, or sold in the formof a discrete solid dose unit including, but not limited to, a tablet, ahard or soft capsule, a cachet, a troche, or a lozenge, each containinga predetermined amount of the active ingredient. Other formulationssuitable for oral administration include, but are not limited to, apowdered or granular formulation, an aqueous or oily suspension, anaqueous or oily solution, or an emulsion.

As used herein, an “oily” liquid is one which comprises acarbon-containing liquid molecule and which exhibits a less polarcharacter than water.

A tablet comprising the active ingredient may, for example, be made bycompressing or molding the active ingredient, optionally with one ormore additional ingredients. Compressed tablets may be prepared bycompressing, in a suitable device, the active ingredient in afree-flowing form such as a powder or granular preparation, optionallymixed with one or more of a binder, a lubricant, an excipient, a surfaceactive agent, and a dispersing agent. Molded tablets may be made bymolding, in a suitable device, a mixture of the active ingredient, apharmaceutically acceptable carrier, and at least sufficient liquid tomoisten the mixture. Pharmaceutically acceptable excipients used in themanufacture of tablets include, but are not limited to, inert diluents,granulating and disintegrating agents, binding agents, and lubricatingagents. Known dispersing agents include, but are not limited to, potatostarch and sodium starch glycollate. Known surface active agentsinclude, but are not limited to, sodium lauryl sulphate. Known diluentsinclude, but are not limited to, calcium carbonate, sodium carbonate,lactose, microcrystalline cellulose, calcium phosphate, calcium hydrogenphosphate, and sodium phosphate. Known granulating and disintegratingagents include, but are not limited to, corn starch and alginic acid.Known binding agents include, but are not limited to, gelatin, acacia,pre-gelatinized maize starch, polyvinylpyrrolidone, and hydroxypropylmethylcellulose. Known lubricating agents include, but are not limitedto, magnesium stearate, stearic acid, silica, and talc.

Tablets may be non-coated or may be coated using known methods toachieve delayed disintegration in the gastrointestinal tract of asubject, thereby providing sustained release and absorption of theactive ingredient. By way of example, a material such as glycerylmonostearate or glyceryl distearate may be used to coat tablets. Furtherby way of example, tablets may be coated using methods described in U.S.Pat. Nos. 4,256,108; 4,160,452; and U.S. Pat. No. 4,265,874 to formosmotically-controlled release tablets. Tablets may further comprise asweetening agent, a flavoring agent, a coloring agent, a preservative,or some combination of these in order to provide pharmaceuticallyelegant and palatable preparation.

Hard capsules comprising the active ingredient may be made using aphysiologically degradable composition, such as gelatin. Such hardcapsules comprise the active ingredient, and may further compriseadditional ingredients including, for example, an inert solid diluentsuch as calcium carbonate, calcium phosphate, or kaolin.

Soft gelatin capsules comprising the active ingredient may be made usinga physiologically degradable composition, such as gelatin. Such softcapsules comprise the active ingredient, which may be mixed with wateror an oil medium such as peanut oil, liquid paraffin, or olive oil.

Lactulose can also be used as a freely erodible filler and is usefulwhen the compounds of the invention are prepared in capsule form.

Liquid formulations of a pharmaceutical composition of the inventionwhich are suitable for oral administration may be prepared, packaged,and sold either in liquid form or in the form of a dry product intendedfor reconstitution with water or another suitable vehicle prior to use.

Liquid suspensions may be prepared using conventional methods to achievesuspension of the active ingredient in an aqueous or oily vehicle.Aqueous vehicles include, for example, water and isotonic saline. Oilyvehicles include, for example, almond oil, oily esters, ethyl alcohol,vegetable oils such as arachis, olive, sesame, or coconut oil,fractionated vegetable oils, and mineral oils such as liquid paraffin.Liquid suspensions may further comprise one or more additionalingredients including, but not limited to, suspending agents, dispersingor wetting agents, emulsifying agents, demulcents, preservatives,buffers, salts, flavorings, coloring agents, and sweetening agents. Oilysuspensions may further comprise a thickening agent. Known suspendingagents include, but are not limited to, sorbitol syrup, hydrogenatededible fats, sodium alginate, polyvinylpyrrolidone, gum tragacanth, gumacacia, and cellulose derivatives such as sodium carboxymethylcellulose,methylcellulose, and hydroxypropylmethylcellulose. Known dispersing orwetting agents include, but are not limited to, naturally occurringphosphatides such as lecithin, condensation products of an alkyleneoxide with a fatty acid, with a long chain aliphatic alcohol, with apartial ester derived from a fatty acid and a hexitol, or with a partialester derived from a fatty acid and a hexitol anhydride (e.g.,polyoxyethylene stearate, heptadecaethyleneoxycetanol, polyoxyethylenesorbitol monooleate, and polyoxyethylene sorbitan monooleate,respectively). Known emulsifying agents include, but are not limited to,lecithin and acacia. Known preservatives include, but are not limitedto, methyl, ethyl, or n-propyl para hydroxybenzoates, ascorbic acid, andsorbic acid. Known sweetening agents include, for example, glycerol,propylene glycol, sorbitol, sucrose, and saccharin. Known thickeningagents for oily suspensions include, for example, beeswax, hardparaffin, and cetyl alcohol.

In one aspect, a preparation in the form of a syrup or elixir or foradministration in the form of drops may comprise active ingredientstogether with a sweetener, which can be calorie-free, and which mayfurther include methylparaben or propylparaben as antiseptics, aflavoring and a suitable color.

Liquid solutions of the active ingredient in aqueous or oily solventsmay be prepared in substantially the same manner as liquid suspensions,the primary difference being that the active ingredient is dissolved,rather than suspended in the solvent. Liquid solutions of thepharmaceutical composition of the invention may comprise each of thecomponents described with regard to liquid suspensions, it beingunderstood that suspending agents will not necessarily aid dissolutionof the active ingredient in the solvent. Aqueous solvents include, forexample, water and isotonic saline. Oily solvents include, for example,almond oil, oily esters, ethyl alcohol, vegetable oils such as arachis,olive, sesame, or coconut oil, fractionated vegetable oils, and mineraloils such as liquid paraffin.

Powdered and granular formulations of a pharmaceutical preparation ofthe invention may be prepared using known methods. Such formulations maybe administered directly to a subject, used, for example, to formtablets, to fill capsules, or to prepare an aqueous or oily suspensionor solution by addition of an aqueous or oily vehicle thereto. Each ofthese formulations may further comprise one or more of a dispersing orwetting agent, a suspending agent, and a preservative. Additionalexcipients, such as fillers and sweetening, flavoring, or coloringagents, may also be included in these formulations.

A pharmaceutical composition of the invention may also be prepared,packaged, or sold in the form of oil in water emulsion or a water-in-oilemulsion. The oily phase may be a vegetable oil such as olive or arachisoil, a mineral oil such as liquid paraffin, or a combination of these.Such compositions may further comprise one or more emulsifying agentsincluding naturally occurring gums such as gum acacia or gum tragacanth,naturally occurring phosphatides such as soybean or lecithinphosphatide, esters or partial esters derived from combinations of fattyacids and hexitol anhydrides such as sorbitan monooleate, andcondensation products of such partial esters with ethylene oxide such aspolyoxyethylene sorbitan monooleate. These emulsions may also containadditional ingredients including, for example, sweetening or flavoringagents.

A pharmaceutical composition of the invention may be prepared, packaged,or sold in a formulation suitable for rectal administration. Such acomposition may be in the form of, for example, a suppository, aretention enema preparation, and a solution for rectal or colonicirrigation.

Suppository formulations may be made by combining the active ingredientwith a non irritating pharmaceutically acceptable excipient which issolid at ordinary room temperature (i.e. about 20° C.) and which isliquid at the rectal temperature of the subject (i.e. about 37° C. in ahealthy human). Suitable pharmaceutically acceptable excipients include,but are not limited to, cocoa butter, polyethylene glycols, and variousglycerides. Suppository formulations may further comprise variousadditional ingredients including, but not limited to, antioxidants andpreservatives.

Retention enema preparations or solutions for rectal or colonicirrigation may be made by combining the active ingredient with apharmaceutically acceptable liquid carrier. As is well known in the art,enema preparations may be administered using, and may be packagedwithin, a delivery device adapted to the rectal anatomy of the subject.Enema preparations may further comprise various additional ingredientsincluding, but not limited to, antioxidants and preservatives.

A pharmaceutical composition of the invention may be prepared, packaged,or sold in a formulation suitable for vaginal administration. Such acomposition may be in the form of, for example, a suppository, animpregnated or coated vaginally-insertable material such as a tampon, adouche preparation, or gel or cream or a solution for vaginalirrigation.

Methods for impregnating or coating a material with a chemicalcomposition are known in the art, and include, but are not limited tomethods of depositing or binding a chemical composition onto a surface,methods of incorporating a chemical composition into the structure of amaterial during the synthesis of the material (i.e. such as with aphysiologically degradable material), and methods of absorbing anaqueous or oily solution or suspension into an absorbent material, withor without subsequent drying.

Douche preparations or solutions for vaginal irrigation may be made bycombining the active ingredient with a pharmaceutically acceptableliquid carrier. As is well known in the art, douche preparations may beadministered using, and may be packaged within, a delivery deviceadapted to the vaginal anatomy of the subject. Douche preparations mayfurther comprise various additional ingredients including, but notlimited to, antioxidants, antibiotics, antifungal agents, andpreservatives.

As used herein, “parenteral administration” of a pharmaceuticalcomposition includes any route of administration characterized byphysical breaching of a tissue of a subject and administration of thepharmaceutical composition through the breach in the tissue. Parenteraladministration thus includes, but is not limited to, administration of apharmaceutical composition by injection of the composition, byapplication of the composition through a surgical incision, byapplication of the composition through a tissue-penetrating non-surgicalwound, and the like. In particular, parenteral administration iscontemplated to include, but is not limited to, subcutaneous,intraperitoneal, intramuscular, and intrasternal injection, and kidneydialytic infusion techniques.

Formulations of a pharmaceutical composition suitable for parenteraladministration comprise the active ingredient combined with apharmaceutically acceptable carrier, such as sterile water or sterileisotonic saline. Such formulations may be prepared, packaged, or sold ina form suitable for bolus administration or for continuousadministration. Injectable formulations may be prepared, packaged, orsold in unit dosage form, such as in ampules or in multi-dose containerscontaining a preservative. Formulations for parenteral administrationinclude, but are not limited to, suspensions, solutions, emulsions inoily or aqueous vehicles, pastes, and implantable sustained-release orbiodegradable formulations. Such formulations may further comprise oneor more additional ingredients including, but not limited to,suspending, stabilizing, or dispersing agents. In one embodiment of aformulation for parenteral administration, the active ingredient isprovided in dry (i.e., powder or granular) form for reconstitution witha suitable vehicle (e.g., sterile pyrogen free water) prior toparenteral administration of the reconstituted composition.

The pharmaceutical compositions may be prepared, packaged, or sold inthe form of a sterile injectable aqueous or oily suspension or solution.This suspension or solution may be formulated according to the knownart, and may comprise, in addition to the active ingredient, additionalingredients such as the dispersing agents, wetting agents, or suspendingagents described herein. Such sterile injectable formulations may beprepared using a non-toxic parenterally acceptable diluent or solvent,such as water or 1,3-butane diol, for example. Other acceptable diluentsand solvents include, but are not limited to, Ringer's solution,isotonic sodium chloride solution, and fixed oils such as syntheticmono- or di-glycerides. Other parentally-administrable formulationswhich are useful include those which comprise the active ingredient inmicrocrystalline form, in a liposomal preparation, or as a component ofa biodegradable polymer systems. Compositions for sustained release orimplantation may comprise pharmaceutically acceptable polymeric orhydrophobic materials such as an emulsion, an ion exchange resin, asparingly soluble polymer, or a sparingly soluble salt.

Formulations suitable for topical administration include, but are notlimited to, liquid or semi-liquid preparations such as liniments,lotions, oil in water or water in oil emulsions such as creams,ointments or pastes, and solutions or suspensions.Topically-administrable formulations may, for example, comprise fromabout 1% to about 10% (w/w) active ingredient, although theconcentration of the active ingredient may be as high as the solubilitylimit of the active ingredient in the solvent. Formulations for topicaladministration may further comprise one or more of the additionalingredients described herein.

A pharmaceutical composition of the invention may be prepared, packaged,or sold in a formulation suitable for pulmonary administration via thebuccal cavity. Such a formulation may comprise dry particles whichcomprise the active ingredient and which have a diameter in the rangefrom about 0.5 to about 7 nanometers, and from about 1 to about 6nanometers. Such compositions are conveniently in the form of drypowders for administration using a device comprising a dry powderreservoir to which a stream of propellant may be directed to dispersethe powder or using a self-propelling solvent/powder-dispensingcontainer such as a device comprising the active ingredient dissolved orsuspended in a low-boiling propellant in a sealed container. Suchpowders can comprise particles wherein at least 98% of the particles byweight have a diameter greater than 0.5 nanometers and at least 95% ofthe particles by number have a diameter less than 7 nanometers. In oneembodiment, at least 95% of the particles by weight have a diametergreater than 1 nanometer and at least 90% of the particles by numberhave a diameter less than 6 nanometers. Dry powder compositions caninclude a solid fine powder diluent such as sugar and are convenientlyprovided in a unit dose form.

Low boiling propellants generally include liquid propellants having aboiling point of below 65° F. at atmospheric pressure. Generally, thepropellant may constitute about 50% to about 99.9% (w/w) of thecomposition, and the active ingredient may constitute about 0.1% toabout 20% (w/w) of the composition. The propellant may further compriseadditional ingredients such as a liquid non-ionic or solid anionicsurfactant or a solid diluent (including those having a particle size ofthe same order as particles comprising the active ingredient).

Pharmaceutical compositions of the invention formulated for pulmonarydelivery may also provide the active ingredient in the form of dropletsof a solution or suspension. Such formulations may be prepared,packaged, or sold as aqueous or dilute alcoholic solutions orsuspensions, optionally sterile, comprising the active ingredient, andmay conveniently be administered using any nebulization or atomizationdevice. Such formulations may further comprise one or more additionalingredients including, but not limited to, a flavoring agent such assaccharin sodium, a volatile oil, a buffering agent, a surface activeagent, or a preservative such as methylhydroxybenzoate. The dropletsprovided by this route of administration may have an average diameter inthe range from about 0.1 to about 200 nanometers.

The formulations described herein as being useful for pulmonary deliveryare also useful for intranasal delivery of a pharmaceutical compositionof the invention.

Another formulation suitable for intranasal administration is a coarsepowder comprising the active ingredient and having an average particlefrom about 0.2 to about 500 micrometers. Such a formulation isadministered in the manner in which snuff is taken, i.e., by rapidinhalation through the nasal passage from a container of the powder heldclose to the nares.

Formulations suitable for nasal administration may, for example,comprise from about as little as about 0.1% (w/w) and as much as about100% (w/w) of the active ingredient, and may further comprise one ormore of the additional ingredients described herein.

A pharmaceutical composition of the invention may be prepared, packaged,or sold in a formulation suitable for buccal administration. Suchformulations may, for example, be in the form of tablets or lozengesmade using conventional methods, and may, for example, comprise about0.1% to about 20% (w/w) active ingredient, the balance comprising anorally dissolvable or degradable composition and, optionally, one ormore of the additional ingredients described herein. Alternately,formulations suitable for buccal administration may comprise a powder oran aerosolized or atomized solution or suspension comprising the activeingredient. Such powdered, aerosolized, or atomized formulations, whendispersed, can have an average particle or droplet size in the rangefrom about 0.1 to about 200 nanometers, and may further comprise one ormore of the additional ingredients described herein.

A pharmaceutical composition of the invention may be prepared, packaged,or sold in a formulation suitable for ophthalmic administration. Suchformulations may, for example, be in the form of eye drops including,for example, a 0.10% to 1.0% (w/w) solution or suspension of the activeingredient in an aqueous or oily liquid carrier. Such drops may furthercomprise buffering agents, salts, or one or more other of the additionalingredients described herein. Other opthalmically-administrableformulations which are useful include those which comprise the activeingredient in microcrystalline form or in a liposomal preparation.

A pharmaceutical composition of the invention may be prepared, packaged,or sold in a formulation suitable for intramucosal administration. Thepresent invention provides for intramucosal administration of compoundsto allow passage or absorption of the compounds across mucosa. Such typeof administration is useful for absorption orally (gingival, sublingual,buccal, etc.), rectally, vaginally, pulmonary, nasally, etc.

In some aspects, sublingual administration has an advantage for activeingredients which in some cases, when given orally, are subject to asubstantial first pass metabolism and enzymatic degradation through theliver, resulting in rapid metabolization and a loss of therapeuticactivity related to the activity of the liver enzymes that convert themolecule into inactive metabolites, or the activity of which isdecreased because of this bioconversion.

In some cases, a sublingual route of administration is capable ofproducing a rapid onset of action due to the considerable permeabilityand vascularization of the buccal mucosa. Moreover, sublingualadministration can also allow the administration of active ingredientswhich are not normally absorbed at the level of the stomach mucosa ordigestive mucosa after oral administration, or alternatively which arepartially or completely degraded in acidic medium after ingestion of,for example, a tablet.

Sublingual tablet preparation techniques known from the prior art areusually prepared by direct compression of a mixture of powderscomprising the active ingredient and excipients for compression, such asdiluents, binders, disintegrating agents and adjuvants. In analternative method of preparation, the active ingredient and thecompression excipients can be dry- or wet-granulated beforehand. In oneaspect, the active ingredient is distributed throughout the mass of thetablet. WO 00/16750 describes a tablet for sublingual use thatdisintegrates rapidly and comprises an ordered mixture in which theactive ingredient is in the form of microparticles which adhere to thesurface of water-soluble particles that are substantially greater insize, constituting a support for the active microparticles, thecomposition also comprising a mucoadhesive agent. WO 00/57858 describesa tablet for sublingual use, comprising an active ingredient combinedwith an effervescent system intended to promote absorption, and also apH-modifier.

The compounds of the invention can be prepared in a formulation orpharmaceutical composition appropriate for administration that allows orenhances absorption across mucosa. Mucosal absorption enhancers include,but are not limited to, a bile salt, fatty acid, surfactant, or alcohol.In specific embodiments, the permeation enhancer can be sodium cholate,sodium dodecyl sulphate, sodium deoxycholate, taurodeoxycholate, sodiumglycocholate, dimethylsulfoxide or ethanol. In a further embodiment, acompound of the invention can be formulated with a mucosal penetrationenhancer to facilitate delivery of the compound. The formulation canalso be prepared with pH optimized for solubility, drug stability, andabsorption through mucosa such as nasal mucosa, oral mucosa, vaginalmucosa, respiratory, and intestinal mucosa.

To further enhance mucosal delivery of pharmaceutical agents within theinvention, formulations comprising the active agent may also contain ahydrophilic low molecular weight compound as a base or excipient. Suchhydrophilic low molecular weight compounds provide a passage mediumthrough which a water-soluble active agent, such as a physiologicallyactive peptide or protein, may diffuse through the base to the bodysurface where the active agent is absorbed. The hydrophilic lowmolecular weight compound optionally absorbs moisture from the mucosa orthe administration atmosphere and dissolves the water-soluble activepeptide. The molecular weight of the hydrophilic low molecular weightcompound is generally not more than 10000 and in one embodiment not morethan 3000. Exemplary hydrophilic low molecular weight compounds includepolyol compounds, such as oligo-, di- and monosaccharides such assucrose, mannitol, lactose, L-arabinose, D-erythrose, D-ribose,D-xylose, D-mannose, D-galactose, lactulose, cellobiose, gentibiose,glycerin, and polyethylene glycol. Other examples of hydrophilic lowmolecular weight compounds useful as carriers within the inventioninclude N-methylpyrrolidone, and alcohols (e.g., oligovinyl alcohol,ethanol, ethylene glycol, propylene glycol, etc.). These hydrophilic lowmolecular weight compounds can be used alone or in combination with oneanother or with other active or inactive components of the intranasalformulation.

When a controlled-release pharmaceutical preparation of the presentinvention further contains a hydrophilic base, many options areavailable for inclusion. Hydrophilic polymers such as a polyethyleneglycol and polyvinyl pyrrolidone, sugar alcohols such as D-sorbitol andxylitol, saccharides such as sucrose, maltose, lactulose, D-fructose,dextran, and glucose, surfactants such as polyoxyethylene-hydrogenatedcastor oil, polyoxyethylene polyoxypropylene glycol, and polyoxyethylenesorbitan higher fatty acid esters, salts such as sodium chloride andmagnesium chloride, organic acids such as citric acid and tartaric acid,amino acids such as glycine, beta-alanine, and lysine hydrochloride, andaminosaccharides such as meglumine are given as examples of thehydrophilic base. In one embodiment, polyethylene glycol, sucrose,polyvinyl pyrrolidone and polyethylene glycol can be used. One or acombination of two or more hydrophilic bases can be used in the presentinvention.

The present invention contemplates pulmonary, nasal, or oraladministration through an inhaler. In one embodiment, delivery from aninhaler can be a metered dose.

An inhaler is a device for patient self-administration of at least onecompound of the invention comprising a spray inhaler (e.g., a nasal,oral, or pulmonary spray inhaler) containing an aerosol sprayformulation of at least one compound of the invention and apharmaceutically acceptable dispersant. In one aspect, the device ismetered to disperse an amount of the aerosol formulation by forming aspray that contains a dose of at least one compound of the inventioneffective to treat a disease or disorder encompassed by the invention.The dispersant may be a surfactant, such as, but not limited to,polyoxyethylene fatty acid esters, polyoxyethylene fatty acid alcohols,and polyoxyethylene sorbitan fatty acid esters. Phospholipid-basedsurfactants also may be used.

In other embodiments, the aerosol formulation is provided as a drypowder aerosol formulation in which a compound of the invention ispresent as a finely divided powder. The dry powder formulation canfurther comprise a bulking agent, such as, but not limited to, lactose,sorbitol, sucrose, and mannitol.

In another specific embodiment, the aerosol formulation is a liquidaerosol formulation further comprising a pharmaceutically acceptablediluent, such as, but not limited to, sterile water, saline, bufferedsaline and dextrose solution.

In further embodiments, the aerosol formulation further comprises atleast one additional compound of the invention in a concentration suchthat the metered amount of the aerosol formulation dispersed by thedevice contains a dose of the additional compound in a metered amountthat is effective to ameliorate the symptoms of disease or disorderdisclosed herein when used in combination with at least a first orsecond compound of the invention.

Thus, the invention provides a self administration method for outpatienttreatment of an addiction related disease or disorder such as analcohol-related disease or disorder. Such administration may be used ina hospital, in a medical office, or outside a hospital or medical officeby non-medical personnel for self administration.

Compounds of the invention will be prepared in a formulation orpharmaceutical composition appropriate for nasal administration. In afurther embodiment, the compounds of the invention can be formulatedwith a mucosal penetration enhancer to facilitate delivery of the drug.The formulation can also be prepared with pH optimized for solubility,drug stability, absorption through nasal mucosa, and otherconsiderations.

Capsules, blisters, and cartridges for use in an inhaler or insufflatormay be formulated to contain a powder mix of the pharmaceuticalcompositions provided herein; a suitable powder base, such as lactose orstarch; and a performance modifier, such as 1-leucine, mannitol, ormagnesium stearate. The lactose may be anhydrous or in the form of themonohydrate. Other suitable excipients include dextran, glucose,maltose, sorbitol, xylitol, fructose, sucrose, and trehalose. Thepharmaceutical compositions provided herein for inhaled/intranasaladministration may further comprise a suitable flavor, such as mentholand levomenthol, or sweeteners, such as saccharin or saccharin sodium.

For administration by inhalation, the compounds for use according to themethods of the invention are conveniently delivered in the form of anaerosol spray presentation from pressurized packs or a nebulizer, withthe use of a suitable propellant, e.g., dichlorodifluoromethane,trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide orother suitable gas. In the case of a pressurized aerosol, the dosageunit may be determined by providing a valve to deliver a metered amount.Capsules and cartridges of e.g., gelatin for use in an inhaler orinsufflator may be formulated containing a powder mix of the drugs and asuitable powder base such as lactose or starch.

As used herein, “additional ingredients” include, but are not limitedto, one or more of the following: excipients; surface active agents;dispersing agents; inert diluents; granulating and disintegratingagents; binding agents; lubricating agents; sweetening agents; flavoringagents; coloring agents; preservatives; physiologically degradablecompositions such as gelatin; aqueous vehicles and solvents; oilyvehicles and solvents; suspending agents; dispersing or wetting agents;emulsifying agents, demulcents; buffers; salts; thickening agents;fillers; emulsifying agents; antioxidants; antibiotics; antifungalagents; stabilizing agents; and pharmaceutically acceptable polymeric orhydrophobic materials. Other “additional ingredients” which may beincluded in the pharmaceutical compositions of the invention are knownin the art and described, for example in Genaro, ed., 1985, Remington'sPharmaceutical Sciences, Mack Publishing Co., Easton, Pa., which isincorporated herein by reference.

Typically, dosages of the compounds of the invention which may beadministered to an animal, including a human, range in amount from about1.0 μg to about 100 g per kilogram of body weight of the animal. Theprecise dosage administered will vary depending upon any number offactors, including but not limited to, the type of animal and type ofdisease state being treated, the age of the animal and the route ofadministration. In one embodiment, the dosage of the compound will varyfrom about 1 mg to about 10 g per kilogram of body weight of the animal.In another embodiment, the dosage will vary from about 10 mg to about 1g per kilogram of body weight of the animal.

The compounds may be administered to a subject as frequently as severaltimes daily, or it may be administered less frequently, such as once aday, once a week, once every two weeks, once a month, or even lessfrequently, such as once every several months or even once a year orless. The frequency of the dose will be readily apparent to the skilledartisan and will depend upon any number of factors, such as, but notlimited to, the type and severity of the disease being treated, the typeand age of the animal, etc.

The invention also includes a kit comprising the compounds of theinvention and an instructional material that describes administration ofthe compounds. In another embodiment, this kit comprises a (e.g.,sterile) solvent suitable for dissolving or suspending the compositionof the invention prior to administering the compound to the mammal.

As used herein, an “instructional material” includes a publication, arecording, a diagram, or any other medium of expression that can be usedto communicate the usefulness of the compounds of the invention in thekit for effecting alleviation of the various diseases or disordersrecited herein. Optionally, or alternately, the instructional materialmay describe one or more methods of alleviating the diseases ordisorders. The instructional material of the kit of the invention may,for example, be affixed to a container that contains a compound of theinvention or be shipped together with a container that contains thecompounds. Alternatively, the instructional material may be shippedseparately from the container with the intention that the instructionalmaterial and the compound be used cooperatively by the recipient.

Other methods and techniques useful for the practice of the inventionthat are not described are known in the art, for example, seeInternational application no. PCT/US2008/064232.

Without further description, it is believed that one of ordinary skillin the art can, using the preceding description and the followingillustrative examples, make and utilize the compounds of the presentinvention and practice the claimed methods. The following workingexamples, therefore, point out embodiments of the present invention, andare not to be construed as limiting in any way the remainder of thedisclosure.

EXAMPLES

Methamphetamine addiction poses a serious public health problem forwhich there is no approved medication. Topiramate a seizure medicationhas shown efficacy in alcohol, and cocaine addictions. The Phase IIdouble-blind placebo-controlled trial proof of concept study, and othersdisclosed below, were conducted to test the safety and efficacy oftopiramate for the treatment of methamphetamine addiction.

Other methods not described herein are useful for the practice of thepresent invention. For example, see International Patent ApplicationNos.

PCT/US2007/088100 (B. Johnson and N. Ait-Daoud Tiouririne),PCT/US2009/035420 (B. Johnson), and PCT/US2008/064232 (Johnson et al.),all of which are incorporated by reference herein in their entirety.

Example 1

The present application provides compositions and methods useful forsingle drug and combination therapies utilizing ondansetron andtopiramate, in combination with the use of genetic diagnostic assays tohelp determine which treatments will be the best for alcohol or drugdependent subjects.

In PCT/US2009/035420 it was predicted that certain allelic types wouldoffer the best treatment outcome with ondansetron. Based on the genotypematrix below, the present predication suggests that all the cells markedwith an X will be efficacious, with XA being the very best, Y mayrespond somewhat, and Z will not respond or worsen.

TT TG GG LL XA X X LS X Y Y SS X Z Z

Therefore, alcoholics who are in the XA or X cells will have an addedeffect of ondansetron over and above their response to topiramate. Thatis, the combination of ondansetron and topiramate will be moreefficacious than either alone or placebo in individuals with LL/TT,LL/TG, LL/GG, LS/TT, or SS/TT genotype. To that end, the presentdisclosure suggests that alcoholics in the XA, X, or Y cells will havean added effect of ondansetron over and above their response totopiramate. That is, the combination of ondansetron and topiramate willbe more efficacious than either alone or placebo in individuals withLL/TT, LL/TG, LL/GG, LS/TT, LS/TG, LS/GG, or SS/TT genotype.

The present disclosure further suggests that alcoholics in the ZZ cellswho receive topiramate will not have an added effect of ondansetron.That is, alcoholics with SS/TG or SS/GG genotype. The present disclosurealso suggests that alcoholics in the Z or Y cells who receive topiramatewould not have an added effect of ondansetron. That is, alcoholics withLS/TG, LS/GG, SS, TG, or SS/GG genotype.

New data supporting these suggestions are provided in the Examplesbelow, show an effect of LL and also LL/TT having a superior response toondansetron compared with placebo. The methods and results of U.S.Provisional Application No. 61/263,599 (B. Johnson), filed Nov. 23,2009, are incorporated by reference herein.

The data further suggest that some individuals with certainpolymorphisms in the glutamate system might not respond to topiramate,thereby diminishing the efficacy of the combination of ondansetron andtopiramate or might experience significant side-effects from topiramatewhich would reduce efficacy of the COMBO because they might take a lowerdose, skip doses, or not take the medication(s) altogether. For example,these polymorphisms might lie in the GluR1-GluR5 system or the GABA-A orGABA-B system.

Useful dosages and combinations include those found in PCT/US2007/088100(B. Johnson and N. Ait-Daoud Tiouririne).

Example 2—Alcohol Dependent Individuals with the TT Vs. (TT/GG) Genotypeof the 3-UTR Region May Respond Differentially to Ondansetron Vs PlaceboTreatment

Two hundred and seventy eight male and female alcohol dependentindividuals were entered into a randomized, double-blind,placebo-controlled, 12-week clinical trial. These individuals weredivided into TT vs. TG/GG) groups of the rs1042173 SNP of the 3-UTR inthe serotonin transporter gene as well as ondansetron (4 meg/kg) vs.placebo groups. The polymorphisms in 3′-UTR region of a gene may affectmRNA expression levels by altering their stability. We propose that theTT genotype of rs1042173 will be associated with lower levels of mRNAexpression compared with those of the TG/GG genotype. We hypothesizethat due to pre-synaptic inhibition at the serotonin autoreceptor, thenet effect of reduced mRNA expression would be a relativehypo-serotonergic state that would lead to upregulated post-synapticserotonin receptors. We, therefore, propose further that ondansetron (aserotonin-3 receptor antagonist) might exert a therapeutic effect inindividuals with the TT genotype by blocking these upregulatedpost-synaptic receptors.

Data was transformed to its natural log to correct for skewness. Mixedmodel analysis of Log Drinks/Drinking day (our primary endpoint tomeasure severity of drinking) data showed that ondansetron is morebeneficial than placebo in reducing severe drinking among individualswith TT genotype (p=0.028). Furthermore, we found that the lowestdrinking was found in the TT group that received ondansetron (TT ond−Logmean=1.36±0.10 Drinks/Drinking Day). In contrast, the TT group thatreceived placebo (TT placebo) consumed a Log mean of 1.61±0.09Drinks/Drinking Day. There was a statistically significant differencebetween the TTond and TG/GG ond groups (Log mean difference=−0.33 95% CI−0.53 to −0.13, p=0.002). From the diagram (FIG. 1 ), it is evident thatthe TT placebo group's drinking was lower than either the TG/GG placebogroup−Log mean=1.72±0.07 Drinks/Drinking Day or the TT/GG ond group−Logmean=1.68±0.07 Drinks/Drinking Day). Furthermore, among placebo, therewas no significant difference in drinking severity between the TT andTG/GG groups (Log mean difference −0.10 95% CI −0.30 to 0.10Drinks/Drinking Day; p=0.315) (FIG. 1 ).

In summary, these data showed that individuals with the TT genotype whoreceived ondansetron improved their drinking outcomes significantly(i.e., show a greater reduction in severe drinking) compared with theirTG/GG counterparts. Also, those with TT genotype who receivedondansetron improved their drinking outcomes significantly compared withindividuals of TT genotype who got placebo. These data, providepreliminary support that ondansetron might be differentially efficaciousamong individuals with the TT genotype.

Example 3—Effects of 5HT3a and 5HT3b SNPs on Ondansetron

Treatment Outcome

The genetic effects of SNPs within the two genes that code for the twosubunits of 5HT3 receptor, on ondansetron treatment outcome wereanalyzed using a mixed-effect linear regression model. In total, tenSNPs in 5HT3a and nine SNPs in 5HT3b genes were analyzed in this study:

Summary of Results for 5HT3b SNP Analysis:

-   -   2 SNPs in the 5HT3b gene (rs4938056 and rs17614942) interacted        significantly with ondansetron treatment:        -   1. rs4938056*treatment: P=0.024; F=5.11 (Both treatment and            genotype main effects were not significant in the population            studied)        -   2. rs17614942*treatment: P=0.01; F=6.62 (In the population            studied, the main effect of treatment was significant at            P=0.019; the genotype main effect was not significant).

The locations of these 2 SNPs within 5HT3b gene are shown in FIG. 2 .

-   -   Ondansetron reduced DDD differentially in the 2 genotypic groups        of rs4938056: T-carriers had a reduction of DDD by 1.30 sd        compared with CC subjects (P=0.013; 95% CI=−2.32 to −0.27).    -   The reduction of DDD by ondansetron was also significantly        different in subjects carrying AC/AA and CC genotypes for        rs17614942: AC/AA treated with ondansetron had a 1.97 sd        reduction in DDD compared to CC subjects.    -   Within the AC/AA group of rs17614942, ondansetron reduced DDD by        2.41 sd compared to the placebo (P=0.008; 95% CI=−4.21 to −0.62)    -   However, within the cohort studied for 5HT3b associations,        ondansetron had a significant effect on reducing DDD, regardless        of the genotypes of rs17614942 (Estimated mean DDD difference        for OND vs. placebo=−1.15 (P=0.019; 95% CI=−2.11 to −0.19)).

FIGS. 3 and 4 shows the DDD in different genotypic groups of rs4938056and rs17614942, respectively.

Summary of Results for 5HT3b SNPs+5HTTLPR Analysis:

rs4938056+5HTTLPR

-   -   The combined genotypes had a slightly greater effect than the        effects of rs4938056 genotypes alone.    -   Ondansetron reduced DDD by 1.82 sd in subjects carrying LL+CT/TT        genotypes compared to subjects who do not carry this genotype        combination (P=0.001; 95% CI=−2.88 to −0.77).    -   Within LL+CT/TT group, ondansetron reduced DDD significantly        better than placebo (Least square Mean difference=1.74 sd;        P=0.011; 95% CI=−3.08 to −0.04)

rs17614942+5HTTLPR

-   -   When rs17614942 genotypes were combined with 5HTTLPR genotypes        of serotonin transporter gene, ondansetron showed the highest        effect on reducing DDD. Ondansetron reduced DDD by 3.33 sd in        subjects carrying LL+AC/AA genotypes compared to subjects who do        not carry this genotype combination (P=0.001; 95% CI=−5.34 to        −1.32).    -   Within LL+AC/AA group, ondansetron reduced DDD significantly        better than placebo (Least square Mean difference=3.6 sd;        P=0.013; 95% CI=−6.42 to −0.77)

Summary of results for 5HT3a SNP analysis:

SNP rs1062613 in 5HT3a gene showed a marginally significant interactionwith treatment in reducing DDD: rs1062613*treatment: P=0.047; F=3.93(Both treatment and genotype main effects were not significant in thepopulation studied)

-   -   Ondansetron reduced DDD differentially in the 2 genotypic groups        of rs1062613: C-carriers had a reduction of DDD by 1.17 sd        compared with TT subjects (P=0.016; 95% CI=−2.12 to −0.22).

The SNP rs1150226 in 5HT3a also showed a significant association withDDD but its interaction with treatment showed only a trend (P=0.065;F=3.42).

-   -   Ondansetron reduced DDD by 1.74 sd in AG/AA subjects of        rs1150226, compared to GG subjects (P=0.006; 95% CI=−2.97 to        −0.5).    -   Within the AG/AA group of rs1150226, ondansetron reduced DDD by        1.57 sd compared to the placebo (P=0.050; 95% CI=−3.14 to        −0.001)

None of the above 5HT3a SNPs were significant when combined with5-HTTLPR genotypes.

Example 4—Topiramate for the Treatment of Methamphetamine Addiction

Methods—The study was conducted at 8 sites, 140 subjects consented,screened for study eligibility and met DSM-IV criteria formethamphetamine dependence. Subjects were randomized to topiramate orplacebo at 50 mg, escalating to 200 mg topiramate daily over weeks 1-5,and 200 mg daily over weeks 6-12. The primary outcome measure wasabstinence from methamphetamine during study weeks 6 through 12.Methamphetamine use was determined using qualitative urine testing at acentral laboratory. The primary outcome was analyzed using a GeneralizedEstimating Equation (GEE) model during weeks 6 through 12. Secondaryoutcomes included use reduction compared to baseline, relapse rate forsubjects with negative urine at randomization, and craving. Bloodsamples were collected for genetic markers to examine the effects oftopiramate on gene expression and to correlate genetic subtypes withresponse to treatment. RNA samples were collected from whole blood for50 topiramate- and 49 placebo-treated individuals at baseline and weeks8 and 12. Genome-wide expression profiles between positive and negativeresponders for Weeks 8 and 12 from placebo- and topiramate-treatedgroups were compared.

Results-(see FIGS. 5-9 ) 338 adults ages 18-45 were screened in order toenroll 140 subjects at eight sites. Of these, 77 (55%) subjectscompleted the 12 weeks of treatment and at least one visit in week 13.For weeks 6-12 partial urinalysis data was available on 105 subjects,with 66 subjects providing samples during week 12. The mean age was 38.4years and 37.5 years in the topiramate group the placebo grouprespectively, males comprised 59.4% of the sample in the topiramategroup, compared to (67.6%) taking placebo. Topiramate was generallywell-tolerated and safe.

For the primary outcome variable, no significant treatment effect fortopiramate was seen. In general, the results were similar for othersecondary outcomes derived directly from the urinalysis data.Exploratory analyses of the data, however, indicated that subjects(n=35) whose baseline use was <18 days out of the previous 30, or whohad negative urine prior to randomization (n=26) had a significanttreatment effect on topiramate (p=0.03 and 0.02 respectively).

At the single-gene level, 1848, 959, 675, and 741 differentiallyexpressed genes were identified, respectively, for Week 8 topiramate,Week 8 placebo, Week 12 topiramate, and Week 12 placebo groups. Amongthem, some genes involved in nervous system development, metabolism orother fundamental functions were only shared by Weeks 8 and 12topiramate groups: PRKACB, USP38, BUB3, GPR183, GNG2, PTPRN2, USP16,ZNF443, ACAA2, SLC30A6, UBE2A were all up-regulated and that CAMTA2,SLCO3A1, TIMP2, SLC19A1, ADM, TLR5, and NTNG2 were down-regulated, inthe topiramate-treated groups. Pathway analysis revealed that 24enriched pathways were shared between Weeks 8 and 12 topiramate groups.Six biological pathways were detected by at least two pathway discoverytools; four of these pathways, long-term potentiation, Fc epsilon RIsignaling pathway, MAPK signaling pathway and GnRH signaling pathway,have been previously reported to play roles in drug addiction.

Conclusions—Example 4—Limitations of the study were the high attritionrate by the end of the study and underestimation of the minimumeffective dose. The majority of subjects also failed to reach a dailydose of at least 150 mg of topiramate, and only six subjects reportedtaking 200 mg daily. Topiramate significantly modulated the expressionlevels of genes involved in several biological processes underlyingaddiction behavior, but insufficient numbers of subjects at week 12prevent definitive correlations between treatment with topiramate andsubsequent genetic modulation. Despite the failure of the per protocoloutcome variables a subset of “lighter” methamphetamine users wereidentified as positive responders to treatment. Future investigationswith topiramate should concentrate on reaching a dose of at least 150mg/day and on identifying patients who are amenable to initiatingabstinence and investigating topiramate's role as a relapse preventionmedication.

Example 5

-   -   1. RS1062613 in 5-HT3A        -   a. RS1062613 SNP

TABLE 1 Example 5: ANOVA of Drinks per Drinking Days Drinks per DrinkingDays Variable F-Value P-Value RS1062613 (TT, TC/CC) 1.64 0.201 Treatment0.67 0.412 RS1062613*Treatment 3.23 0.072

TABLE 2 Example 5: Least Squares Mean Effect Estimated Mean StandardError TC/CC 5.58 0.29 TT 6.02 0.25 Placebo 5.94 0.27 OND 5.66 0.27TC/CC: Placebo 6.03 0.39 OND 5.13 0.40 TT: Placebo 5.85 0.34 OND 6.190.33

TABLE 3 Example 5: Least Squares Mean Difference between Treatment andPlacebo and Its 95% Confidence Intervals Estimated Mean Lower 95% Upper95% Effect Difference C.I. C.I. P-Value Among OND: TC/CC vs. TT −1.06−2.01 −0.11 0.029 Among TC/CC: OND vs. Placebo −0.90 −1.94 0.14 0.089

TABLE 4 Example 5: Frequency of RS1062613 RS1062613 Frequency CC 13(4.4%) TC 107 (36.2%) TT 176 (59.5%)

-   -   -   b. 5-HTTLPR and RS1062613

TABLE 1 Part b. - Example 5: ANOVA of Drinks per Drinking Days Drinksper Drinking Days Variable F-Value P-Value RS1062613 (TT, TC/CC) 1.750.186 Treatment 2.03 0.154 RS1062613*Treatment 3.02 0.082 5-HTTLPR (LL,LS/SS) 2.94 0.086 5-HTTLPR*Treatment. 4.79 0.029

TABLE 2 Part b. Example 5: Least Squares Mean Effect Estimated MeanStandard Error TC/CC 5.53 0.30 TT 5.98 0.26 Placebo 6.01 0.29 OND 5.500.28 TC/CC: Placebo 6.08 0.40 OND 4.98 0.40 TT: Placebo 5.94 0.36 OND6.02 0.33 LS/SS 6.06 0.25 LL 5.45 0.31 LS/SS: Placebo 5.93 0.32 OND 6.190.33 LL: Placebo 6.09 0.45 OND 4.80 0.41

TABLE 3 Part b, Example 5: Least Squares Mean Difference betweenTreatment and Placebo and Its 95% Confidence Intervals Estimated MeanLower 95% Upper 95% Effect Difference C.I. C.I. P-Value Among OND: LLvs. LS/SS −1.39 −2.36 −0.42 0.005 Among LL: OND vs. Placebo −1.29 −2.43−0.15 0.027 Among OND: TC/CC vs. TT −1.05 −1.99 −0.10 0.030 Among TC/CC:OND vs. Placebo −1.10 −2.15 −0.06 0.039

-   -   2. RS1150226 in 5-HT3A        -   a. RS1150226

TABLE 1 Section 2, Part a, Example 5: ANOVA of Drinks per Drinking DaysDrinks per Drinking Days Variable F-Value P-Value RS150226 (GG, AG/AA)3.17 0.075 Treatment 1.93 0.165 RS1150226*Treatment 2.35 0.126

TABLE 2 Section 2, Part a, Example 5: Least Squares Mean EffectEstimated Mean Standard Error AG/AA 5.22 0.41 GG 6.01 0.23 Placebo 5.920.33 OND 5.31 0.33 AG/AA: Placebo 5.86 0.58 OND 4.58 0.58 GG: Placebo5.98 0.30 OND 6.04 0.29

TABLE 3 Section 2, Part a: Least Squares Mean Difference betweenTreatment and Placebo and Its 95% Confidence Intervals Estimated MeanLower 95% Upper 95% Effect Difference C.I. C.I. P-Value Among OND: AG/AAvs. GG −1.47 −2.69 −0.24 0.019 AG/AA vs. GG −0.79 −1.66 0.08 0.075

TABLE 4 Section 2, Part a, Example 5: Frequency of RS1150226 RS1150226Frequency AA 5 (1.7%) AG 47 (15.9%) GG 243 (82.4%)

-   -   -   b. 5-HTTLPR and RS1150226

TABLE 1 Section 2, Part b, Example 5: ANOVA of Drinks per Drinking DaysDrinks per Drinking Days Variable F-Value P-Value RS1150226 (GG, AG/AA)2.48 0.116 Treatment 2.79 0.095 RS1150226*Treatment 1.54 0.214 5-HTTLPR(LL, LS/SS) 2.34 0.126 5-HTTLPR*Treatment 4.02 0.045

TABLE 2 Section 2, Part b, Example 5: Least Squares Mean EffectEstimated Mean Standard Error AG/AA 5.25 0.41 GG 5.95 0.24 Placebo 5.970.34 OND 5.23 0.33 AG/AA: Placebo 5.90 0.58 OND 4.60 0.57 GG: Placebo6.05 0.31 OND 5.85 0.30 LS/SS 5.87 0.29 LL 5.33 0.33 LS/SS: Placebo 5.890.37 OND 5.86 0.40 LL: Placebo 6.06 0.48 OND 4.59 0.43

TABLE 3 Section 2, Part b, Example 5: Least Squares Mean Differencebetween Treatment and Placebo and Its 95% Confidence Intervals EstimatedMean Lower 95% Upper 95% Effect Difference C.I. C.I. P-Value Among OND;LL vs. LS/SS −1.27 −2.25 −0.29 0.011 Among LL: OND vs. Placebo −1.47−2.70 −0.23 0.020 Among OND: AG/AA vs. GG −1.25 −2.48 −0.01 0.047

-   -   3. RS17614942 in 5-H T3B        -   a. RS17614942

TABLE 1 Section 3, Part a, Example 5: ANOVA of Drinks per Drinking DaysDrinks per Drinking Days Variable F-Value P-Value RS17614942 (CC, AC/AA)1.09 0.297 Treatment 3.86 0.049 RS17614942*Treatment 4.85 0.028

TABLE 2 Section 3, Part a, Example 5: Least Squares Mean EffectEstimated Mean Standard Error AC/AA 5.42 0.47 CC 5.94 0.22 Placebo 6.160.37 OND 5.20 0.37 AC/AA: Placebo 6.44 0.66 OND 4.40 0.66 CC: Placebo5.88 0.29 OND 6.00 0.29

TABLE 3 Section 3, Part a, Example 5: Least Squares Mean Differencebetween Treatment and Placebo and Its 95% Confidence Intervals EstimatedMean Lower 95% Upper 95% Effect Difference C.I. C.I. P-Value Among OND:AC/AA vs. CC −1.60 −3.00 −0.23 0.022 Among AC/AA: OND vs. Placebo −2.04−3.83 −0.26 0.025 OND vs. Placebo −0.96 −1.92 −0.002 0.050

TABLE 4 Section 3, Part a, Example 5: Frequency of RS17614942 RS17614942Frequency AA 2 (0.7%) AC 38 (12.9%) CC 255 (86.4%)

-   -   -   b. RS17614942 and 5-HTTLPR

TABLE 1 Section 3, Part b, Example 5: ANOVA of Drinks per Drinking DaysDrinks per Drinking Days Variable F-Value P-Value RS17614942 (CC, AC/AA)0.83 0.362 5-HTTLPR (LL, LS/SS) 2.77 0.096 Treatment 5.17 0.023RS17614942*Treatment 4.23 0.040 5-HTTLPR*Treatment 4.23 0.040

TABLE 2 Section 3, Part b, Example 5: Least Squares Mean EffectEstimated Mean Standard Error AC/AA 5.43 0.47 CC 5.88 0.23 Placebo 6.220.38 OND 5.09 0.37 AC/AA: Placebo 6.50 0.65 OND 4.37 0.65 CC: Placebo5.94 0.31 OND 5.82 0.29 LS/SS 5.95 0.31 LL 5.36 0.35 LS/SS: Placebo 6.150.41 OND 5.76 0.42 LL: Placebo 6.29 0.50 OND 4.43 0.46

TABLE 3 Section 3, Part b, Example 5: Least Squares Mean Differencebetween Treatment and Placebo and Its 95% Confidence Intervals EstimatedMean Lower 95% Upper 95% Effect Difference C.I. C.I. P-Value OND vs.Placebo −1.13 −2.10 −0.15 0.023 Among OND: LL vs. LS/SS −1.33 −2.30−0.36 0.008 Among LL: OND vs. Placebo −1.86 −3.16 −0.56 0.005 Among OND:AC/AA vs. CC −1.45 −2.81 −0.09 0.037 Among AC/AA: OND vs. Placebo −2.13−3.91 −0.35 0.019

-   -   4. RS4938056 in 5-HT3B        -   a. RS4938056

TABLE 1 Section 4, Part a, Example 5: ANOVA of Drinks per Drinking DaysDrinks per Drinking Days Variable F-Value P-Value RS4938056 (TT/TC, CC)1.51 0.219 Treatment 0.23 0.628 RS4938056*Treatment 4.66 0.031

TABLE 2 Section 4, Part a, Example 5: Least Squares Mean EffectEstimated Mean Standard Error CC 6.19 0.33 CT/TT 5.73 0.24 Placebo 5.870.29 OND 6.05 0.29 CC: Placebo 5.71 0.44 OND 6.67 0.46 CT/TT: Placebo6.03 0.32 OND 5.43 0.31

TABLE 3 Section 4, Part a, Example 5: Least Squares Mean Differencebetween Treatment and Placebo and Its 95% Confidence Intervals EstimatedMean Lower 95% Upper 95% Effect Difference C.I. C.I. P-Value Among OND:CT/TT vs. CC −1.24 −2.27 −0.21 0.018

TABLE 4 Section 4, Part a, Example 5: Frequency of RS4938056 RS4938056Frequency TT 68 (23.4%) CT 131 (45.0%) CC 92 (31.6%)

-   -   -   b. RS4938056 and 5-HTTLPR

TABLE 4 Section 4, Part a, Example 5: Frequency of RS4938056 RS4938056Frequency TT 68 (23.4%) CT 131 (45.0%) CC 92 (31.6%)

TABLE 2 Section 4, Part b, Example 5: Least Squares Mean EffectEstimated Mean Standard Error CC 6.10 0.34 CT/TT 5.70 0.25 Placebo 5.940.30 OND 5.86 0.30 CC: Placebo 5.77 0.45 OND 6.42 0.46 CT/TT: Placebo6.10 0.33 OND 5.29 0.31 LS/SS 6.20 0.25 LL 5.60 0.33 LS/SS: Placebo 5.850.32 OND 6.54 0.34 LL: Placebo 6.02 0.46 OND 5.18 0.43

TABLE 3 Section 4, Part b, Example 5: Least Squares Mean Differencebetween Treatment and Placebo and Its 95% Confidence Intervals EstimatedMean Lower 95% Upper 95% Effect Difference C.I. C.I. P-Value Among OND:LL vs. LS/SS −1.37 −2.34 −0.39 0.006 Among OND: CT/TT vs. CC −1.13 −2.15−0.11 0.031 Among CT/TT OND vs. Placebo −0.81 −1.63 0.01 0.054

Simplified Model

TABLE 1 Section 4, Part c, Example 5: ANOVA of Drinks per Drinking DaysDrinks per Drinking Days Variable F-Value P-Value Comb. Genotype (LL +CT/TT, Others) 2.97 0.085 Treatment 2.28 0.132 Comb. Genotype*Treatment5.87 0.016

TABLE 2 Section 4, Part c, Example 5: Least Squares Mean EffectEstimated Mean Standard Error LL + CT/TT 5.39 0.36 Others 6.07 0.23Placebo 6.02 0.31 OND 5.43 0.30 LL + CT/TT: Placebo 6.16 0.52 OND 4.610.47 Others: Placebo 5.89 0.30 OND 6.25 0.31

TABLE 3 Section 4, Part c, Example 5: Least Squares Mean Differencebetween Treatment and Placebo and Its 95% Confidence Intervals EstimatedMean Lower 95% Upper 95% Effect Difference C.I. C.I. P-Value Among OND:LL + CT/TT vs. −1.64 −2.69 −0.58 0.002 Others LL + CT/TT OND vs. Placebo−1.55 −2.89 −0.20 0.024

TABLE 4 Section 4, Part c, Example 5: Frequency of Combined Genotype andTreatment Combined Genotype Treatment LL and CT/TT Others OND 38 (13.1%)108 (37.1%) Placebo 32 (11.0%) 113 (38.8%)

Example 6—Analysis of Treatment Effects on Usage with Adjustment forDifferences at Baseline Due to Genotype: The Methamphetamine/OndansetronStudy

Methods: Data were modeled using generalized estimating equations (GEE).The dependent variable for analysis was binary: 0=subject usedmethamphetamine during the week and 1=subject did not usemethamphetamine during the week. Independent variables were genotype,group, weeks since randomization and the interaction between treatmentgroup and weeks since randomization. The hypothesis of primary interestasked if treatment with genotype influences the effect of ondansetron onusage. Specifically, this hypothesis was tested by asking if theweek×group×genotype interaction differed significantly from zero. Themodel's group and genotype terms allowed for possible differences atbaseline due to ineffective randomization and genotype, respectively.Group consisted of two levels: placebo vs. all active arms combined,where all active arms were combined at the request of the study sponsorand investigators. Subjects were of three different genotypes-SS, LL andLS. At the request of the investigator, two, separate GEE analyses wererun, one for each pairing of a homozygote with the heterozygote (i.e.,SS-LS and LL-LS). Due to the small quantity of subjects for whichgenotyping was conducted (n=34), hypothesis testing used Type IIIgeneralized score statistics. Analysis was restricted to data obtainedpost-randomization.

Results: Data were available on 34 subjects. The attached resultsindicate that the proportion of subjects with a methamphetamine-freeweek remained roughly steady for the active group but declined over timefor the placebo arm. The difference in change over time between activeand placebo approached statistical significance for the LLLS-adjustedanalysis (p=0.0586) but not for the SSLS-adjusted analysis (p=0.5711).Neither analysis indicates that genotype influenced the treatment effect(p=0.6125 for SSLS and p=0.7740 for LLLS).

All results presented here need to be interpreted with caution, becausedrop-out exceeded 50% by the end of the study (1−15/34=19/34≈0.56), andGEE analysis assumed that drop-outs were not treatment-related.

Proportion with Clean Week SSLS No Yes STUDY WEEK Active Active ELAPSEDSINCE No Yes No Yes RANDOMIZATION N Mean SD N Mean SD N Mean SD N MeanSD N 1 34 0.67 0.58 3 1.00 0.00 2 0.67 0.50 9 0.50 0.51 18 2 30 0.500.71 2 0.50 0.71 2 0.44 0.53 9 0.50 0.52 14 3 29 0.50 0.71 2 1.00 0.00 20.43 0.53 7 0.59 0.51 17 4 28 0.50 0.71 2 0.50 0.71 2 0.25 0.46 8 0.530.52 15 5 23 0.50 0.71 2 0.50 0.71 2 0.33 0.52 6 0.42 0.51 12 6 21 0.500.71 2 0.50 0.71 2 0.33 0.52 6 0.30 0.48 10 7 21 0.00 . 1 0.50 0.71 20.20 0.45 5 0.45 0.52 11 8 15 0.00 0.00 2 0.50 0.71 2 0.00 0.00 4 0.500.55 6

Score Statistics For Type 3 GEE Analysis Chi- Pr > Source DF SquareChiSq Active 1 0.94 0.3323 study_week 1 4.51 0.0337 study_week × Active1 0.32 0.5711 study_week × Active × SSLS 1 0.26 0.6125

Proportion with Clean Week LLLS No Yes STUDY WEEK Active Active ELAPSEDSINCE No Yes No Yes RANDOMIZATION N Mean SD N Mean SD N Mean SD N MeanSD N 1 34 0.67 0.58 3 0.50 0.53 8 0.67 0.50 9 0.58 0.51 12 2 30 0.330.58 3 0.33 0.52 6 0.50 0.53 8 0.60 0.52 10 3 29 0.50 0.71 2 0.71 0.49 70.43 0.53 7 0.58 0.51 12 4 28 0.00 0.00 2 0.50 0.55 6 0.38 0.52 8 0.550.52 11 5 23 0.00 0.00 2 0.25 0.50 4 0.50 0.55 6 0.50 0.53 10 6 21 0.000.00 2 0.50 0.58 4 0.50 0.55 6 0.25 0.46 8 7 21 0.00 0.00 2 0.25 0.50 40.25 0.50 4 0.56 0.53 9 8 15 0.00 0.00 2 0.00 . 1 0.00 0.00 4 0.57 0.537

Score Statistics For Type 3 GEE Analysis Chi- Pr > Source DF SquareChiSq Active 1 0.88 0.3482 study_week 1 4.52 0.0334 study_week × Active1 3.58 0.0586 study_week × Active × LLLS 1 0.08 0.7740

Example 7—Analysis of Treatment Effects on Usage with Adjustment forDifferences at Baseline Due to Genotype: The Methamphetamine/OndansetronStudy (Part II)

Methods: Data were modeled using generalized estimating equations (GEE).The dependent variable for analysis was binary: 0=subject usedmethamphetamine during the week and 1=subject did not usemethamphetamine during the week. Independent variables were genotype,group, weeks since randomization and the interaction between treatmentgroup and weeks since randomization. The hypothesis of primary interestasked if treatment with ondansetron influences rate of change inmethamphetamine usage over time. Specifically, this hypothesis wastested by asking if the week x group interaction differed significantlyfrom zero. The model's group and genotype terms allowed for possibledifferences at baseline due to ineffective randomization and genotype,respectively. Group consisted of two levels: placebo vs. all active armscombined, where all active arms were combined at the request of thestudy sponsor and investigators. Subjects were of three differentgenotypes-SS, LL and LS. At the request of the investigator, two,separate GEE analyses were run, one for each pairing of a homozygotewith the heterozygote (i.e., SS-LS and LL-LS). Due to the small quantityof subjects for which genotyping was conducted (n=34), hypothesistesting used Type III generalized score statistics. Analysis wasrestricted to data obtained post-randomization.

Results: Data were available on 34 subjects. The attached resultsindicate that the proportion of subjects with a methamphetamine-freeweek remained roughly steady for the active group but declined over timefor the placebo arm. The difference in slopes between active and placeboapproached statistical significance for each analysis (p=0.0525 forSSLS-adjusted and p=0.0510 for LLLS-adjusted). These results need to beinterpreted with caution, however, because drop-out exceeded 50% by theend of the study (1-15/34=19/34≈ 0.56), and GEE analysis assumed thatdrop-outs were not treatment-related.

Proportion with Clean Week STUDY WEEK Active Arm ELAPSED SINCE No YesRANDOMIZATION N Mean SD N Mean SD N 1 34 0.67 0.49 12 0.55 0.51 20 2 300.45 0.52 11 0.50 0.52 16 3 29 0.44 0.53 9 0.63 0.50 19 4 28 0.30 0.4810 0.53 0.51 17 5 23 0.38 0.52 8 0.43 0.51 14 6 21 0.38 0.52 8 0.33 0.4912 7 21 0.17 0.41 6 0.46 0.52 13 8 15 0.00 0.00 6 0.50 0.53 8

Chi- p- Source DF Square value SSLS 1 0.55 0.4587 study_week 1 4.550.0329 study_week × Active 1 3.76 0.0525 Active 1 0.68 0.4103

Proportion with Clean Week STUDY WEEK Active Arm ELAPSED SINCE No YesRANDOMIZATION N Mean SD N Mean SD N 1 34 0.67 0.49 12 0.55 0.51 20 2 300.45 0.52 11 0.50 0.52 16 3 29 0.44 0.53 9 0.63 0.50 19 4 28 0.30 0.4810 0.53 0.51 17 5 23 0.38 0.52 8 0.43 0.51 14 6 21 0.38 0.52 8 0.33 0.4912 7 21 0.17 0.41 6 0.46 0.52 13 8 15 0.00 0.00 6 0.50 0.53 8

Chi- p- Source DF Square value LLLS 1 0.34 0.5597 study_week 1 4.580.0323 study_week × Active 1 3.81 0.0510 Active 1 0.83 0.3636

Example 8—Association Between Genotype of the SerotoninTransporter-Linked Polymorphic Region of the Serotonin Transporter Geneand Age of Onset of Methamphetamine Use

Methamphetamine is a highly addictive central nervous system stimulant,and both current and recently abstinent chronicmethamphetamine-dependent individuals can develop irreversiblestructural and neurochemical changes in the brain with long-lastingcognitive and motor deficits (Chang et al., 2002; Seiden and Ricaurte,1987; Thompson et al., 2004).

Methamphetamine dependence is on the rise in the United States and otherparts of the world (Winslow et al., 2007). According to surveys fundedby the National Institute on Drug Abuse in 2005, 10.4 million Americansaged 12 years and older and 4.5% of 12th graders had usedmethamphetamine at least once in their lifetime (National Institute onDrug Abuse, 2006). Increased production and spread of methamphetamineuse to other parts of the country from its traditional endemic areas inthe West and Midwest have raised additional concern about the increasingprevalence of methamphetamine addiction (Ehlers et al., 2007; Johnson etal., in press).

Several studies conducted in various countries, and with differentethnic populations, have reported an increased prevalence of adultmethamphetamine dependence when the onset of methamphetamine useoccurred in adolescence (Nordahl et al., 2003). The progression fromfirst-time drug use to the development of dependence does, however,depend upon the interplay of both genetic and environmental factors(Goldman et al., 2005; McGue et al., 2006); therefore, not alladolescents who experiment with methamphetamine progress tomethamphetamine dependence as adults (Fowler et al., 2007).

The importance of genetic factors has been highlighted by Ehlers andcolleagues (2007), who showed, in a relatively homogenous population ofNative Americans in Southwest California, that the liability toward theinitiation of stimulant use is highly heritable at an estimated rate of38%. Understanding the nature of the genetic factors that increase therisk of stimulant initiation can aid in appropriate screening and earlyintervention for those who are environmentally vulnerable tomethamphetamine exposure, and can facilitate the development of targetedmedications toward the treatment of those who become dependent.

The chronic administration of methamphetamine to rats damages thestructure of the central nervous system by degrading the terminal endsof serotonin (5-HT) neurons (Ricaurte et al., 1980). Human brain imagingstudies also have shown that chronic methamphetamine users can exhibitsignificantly reduced 5-HT transporter (5-HTT) densities, an indicationof terminal neuronal damage, in different brain regions (Sekine et al.,2006). Because these reductions in 5-HTT density occurred in a time- andconcentration-dependent manner, individuals with the earliest onset andgreatest use of methamphetamine can be expected to experience the moststructural brain damage.

5-HT neurons are tonic inhibitors of dopamine neurons in the centralnervous system (Johnson, 2000). Therefore, the degradation of 5-HTneurons can lead to a rise in extracellular dopamine levels (Nordahl etal., 2003), which in turn increases the individual's behavioralpropensity toward further methamphetamine use (Volkow and Li, 2004).

It is, therefore, reasonable to hypothesize that, once established,chronic methamphetamine use sets up a feed-forward process wherebyincreased methamphetamine use leads to a rise in damage to 5-HT neurons,which in turn leads to an enhancement of the behavioral drive to usemore methamphetamine. As a logical extension of this hypothesis, itwould, therefore, be reasonable to suspect that a component of thebiological or genetic vulnerability toward the initiation ofmethamphetamine use might reside within regulatory systems that modulate5-HT function and density.

Of the mechanisms that control synaptic 5-HT function, perhaps the mostcompelling relates to the functional state of the pre-synaptic 5-HTT.The 5-HTT is responsible for removing 5-HT from the synaptic cleft(Lesch et al., 2002). Indeed, up to 60% of neuronal 5-HT function isgated by the 5-HTT. Synaptic clearance of 5-HT is determined by thenumber of 5-HTTs expressed at the pre-synaptic surface and the affinityof 5-HTTs to 5-HT (Beckman and Quick, 1998).

The 5-HTT gene is found at the SLC6A4 locus on chromosome 17q11.1-q12,and its 5′-regulatory promoter region contains a functional polymorphismknown as the 5-HTT-linked polymorphic region (5′-HTTLPR) (Heils et al.,1996, 1997). This polymorphism is an insertion/deletion mutation inwhich the long (L) variant has 44 base pairs that are absent in theshort (S) variant. The L-allelic variant of the 5′-HTTLPR is associatedwith increased transcription rates in lymphoblasts and in cell culture.In the general population, the LL genotype, compared with the SS andheterozygous (LS) genotypes, is associated with greater 5-HT uptake intohuman platelets (Greenberg et al., 1999) and lymphoblasts (Lesch et al.,1996) and greater [¹²³1]2 beta-carboxymethoxy-3beta-(4-iodophenyl)tropane (0-CIT) binding in human raphe nuclei (Heinzet al., 2000). Hence, individuals with the LL genotype have greateruptake and, presumably, reduced intrasynaptic 5-HT levels and 5-HTneurotransmission (Heils et al., 1996; Lesch et al., 1996). Johnson(2000) has proposed that this relative hyposerotonergic state canpredispose an individual to impulsive behavior, includingsubstance-taking behavior. Since heightened levels of impulsive behaviorhave been associated with methamphetamine use (Semple et al., 2005),although it has been debated whether it is a cause or consequence, it ishypothesized that these individuals with the LL genotype may also bemore prone than S-carriers to develop early-onset methamphetamine use.

Early-onset methamphetamine use increases the lifetime prevalence ofmethamphetamine dependence. An earlier onset of methamphetamine useleads to greater damage to the terminal ends of serotonin neurons, morereduction in serotonin transporter (5-HTT) density, and an increasedpropensity toward further methamphetamine use. Because genetic variationwithin the promoter region of the 5 HTT gene (the 5-HTT-linkedpolymorphic region; 5′-HTTLPR) leads to differential expression of the5-HTT, we examined, for the first time, whether this could be acandidate site associated with predisposition toward early-onsetmethamphetamine use. We sought to determine whether there is adifferential association between the long (L) and short (S) polymorphicvariants of the 5′-HTTLPR and the age of first methamphetamine use.

Materials and Methods

Subjects

Thirty-six out of 150 treatment-seeking individuals who consented tothis genetic evaluation, and who were enrolled in a clinical trial forthe treatment of methamphetamine dependence, were included in thisstudy. All subjects were at least 18 years of age and diagnosed asmethamphetamine dependent by Diagnostic and Statistical Manual of MentalDisorders, 4th edition (DSM-IV) criteria (American PsychiatricAssociation, 1994). The enrolled subjects were required to have at leastone methamphetamine-positive urine specimen during the 2-week baselineperiod. They were in good physical health as determined by physical andlaboratory examinations (i.e., hematological assessment, biochemistry,and urinalysis). Exclusion criteria were current dependence on anypsychoactive substance (as defined by DSM-IV criteria) besidesmethamphetamine, nicotine, or marijuana, or physiological dependence onalcohol or a sedative-hypnotic, e.g., a benzodiazepine requiring medicaldetoxification. We also excluded individuals with current diagnoses ofanxiety, affective, or psychotic disorders.

We did not study individuals who: were mandated by the courts to betreated for methamphetamine dependence, were pregnant or not using anacceptable form of contraception (i.e., oral contraceptive, hormonal orsurgical implant, sterilization, or spermicide and barrier), were takingpsychotropic medication, were using opiate substitutes within 2 monthsof enrollment, were asthmatic, or had AIDS.

We received ethics approval from the appropriate institutional reviewboards. Study subjects were recruited between August 2002 and July 2003by newspaper, television, or radio advertisements.

After obtaining written informed consent, and prior to the subjects'enrollment in the clinical trial, we determined psychiatric diagnosisusing the Structured Clinical Interview for DSM-IV (First et al., 1994)and age of onset of methamphetamine use using the Addiction SeverityIndex-Lite (Cacciola et al., 2007). Other structured measures werecollected at enrollment and at scheduled intervals during the clinicaltrial, as reported elsewhere (Johnson et al., in press).

Collection of Blood Samples for Genotyping

Ten milliliters of blood was drawn from each subject at baseline toobtain white blood cells for the determination of 5′-HTTLPR genotypes.

Genotyping

DNA was extracted using a Gentra Puregene® kit (QIAGEN Inc., Valencia,Calif.). Fifty nanograms of genomic DNA was polymerase chain reactionamplified for the 5′-HTTLPR 44-base-pair promoter-region repeatpolymorphism using the primers 5′-TCCTCCG CTTTGGCGCCTCTTCC-3′ (forward;SEQ ID NO:1) and 5′-TGGGG GTTGCAGGGGAGATCCTG-3′ (reverse; SEQ ID NO:2)in a 20-μl final volume with 2.5 U of BIOLASE™ DNA polymerase (Bioline,London, United Kingdom), 1×NH4 reaction buffer, 0.5 mM MgCl2, 0.8 mMdeoxynucleotide triphosphates, dimethyl sulfoxide, and 100 nM of eachprimer. The thermal cycling included initial denaturation at 95° C. for15 min, 45 cycles of 94° C. for 30 s, 65.5° C. for 90 s, and 72° C. for1 min, a final extension of 72° C. for 10 min, and a terminal hold at 4°C. The alleles for the 5′-HTTLPR were separated by gel electrophoresisusing 3% agarose (Cambrex, Rockland, Me.) and visualized by an ethidiumbromide/ultraviolet detection system.

Statistical Analysis

We used the Cox proportional-hazards model to assess the relative riskof an earlier onset of methamphetamine use for the LL genotype comparedwith heterozygotes (LS) and S carriers of the 5′-HTTLPR of the 5-HTTgene. Furthermore, we tested additive, dominant, and recessive geneticmodels in the analyses. We used the Kaplan-Meier method to estimate theprobabilities in time (years) for which individuals with the LL genotypevs. LS heterozygotes or S carriers first used methamphetamine, and alog-rank test to compare these probabilities. An analysis of variancewas used to compare the mean ages of onset of those with the LL genotypevs. LS heterozygotes or S carriers of the 5′-HTTLPR.

RESULTS DNA samples from 36 methamphetamine-dependent subjects agedbetween 19 years and 55 years were genotyped in this study. Of thesesubjects, 78% were White and 12% were Hispanic; 32% were female and 68%male. Additionally, the genotypic distribution of the cohort was 16% LL,53% LS, and 31% SS (Table 1).

The L homozygotes showed a significantly higher risk of an earlier onsetof methamphetamine use compared with LS heterozygotes (hazard ratio=3.7;95% confidence interval [CI]=1.3-10.0; p=0.01). Compared with Shomozygotes, LL subjects also showed an earlier onset of methamphetamineuse (hazard ratio=2.78, 95% CI=0.98-7.69; p=0.05), while the differencebetween LS and SS subjects was not statistically significant. When thecombined mean ages of onset of methamphetamine use in SS and LS subjectswere compared with subjects with the LL genotype, the risk of first-timeuse of methamphetamine in L homozygotes was 3.27 times higher (95%CI=1.26-8.50; p=0.01) than among S-carriers. This suggests a dominanteffect of the L allele over the S allele.

Using the Kaplan-Meier method (FIG. 10 ), it can be seen thatindividuals with the LL genotype compared with their LS or SScounterparts became dependent on methamphetamine significantly earlier(log-rank, all p values<0.05). Furthermore, individuals with the LLgenotype, compared with S-carriers, first used methamphetamine about 5years earlier (p=0.04) (Table 1). Table 1 describes the ages of onset ofmethamphetamine use for the 5′-HTTLPR Genotypes—M=males; F=females.

TABLE 1 Example 8: LL Genotype LS Genotype SS Genotype (n = 6) (n = 19)(n = 11) p Value A. M = 2; F = 4 M = 14; F = 5 M = 2; F = 9 (F Value)Mean (SD) 17.3 (1.63) 22.2 (6.35) 21.9 (3.22) 0.13 (2.11) age of onsetLL Genotype LS/SS Genotype (n = 6) (n = 30) p Value M = 2; F = 4 M = 16;F = 14 (F Value) Mean (SD) 17.3 (1.63) 22.1 (5.35) 0.04 (4.31) age ofonset LL/LS Genotype SS Genotype (n = 25) (n = 11) p Value M = 16; F = 9M = 2; F = 9 (F Value) Mean (SD) 21.0 (5.93) 21.9 (3.22) 0.69 (0.16) ageof onset

Discussion—Example 8

The data disclosed herein suggest that among methamphetamine-dependentindividuals, possession of the LL genotype in the 5′-HTTLPR, comparedwith their S-carrier counterparts, was associated with more than a 3times greater risk of having had an earlier onset of methamphetamineuse.

The 5-HTT plays a role in controlling the duration and degree ofserotonergic neurotransmission (Johnson, 2000). The L allele of thepromoter region of the 5-HTT gene, which transcribes a higher number of5-HTT copies compared with the S allele (Heils et al., 1996), alterstransporter expression levels in different brain regions (Heinz et al.,2000). As mentioned earlier, higher expression levels of 5-HTTs areassociated with increased 5-HT uptake, leading to a relativeintrasynaptic hyposerotonergic state and reduced serotonergicneurotransmission (Greenberg et al., 1999). We also proposed thatindividuals with the LL genotype in the 5′ HTTLPR who possess thisrelative hyposerotonergic state would have a greater propensity towardimpulsive behavior and, consequently, methamphetamine use.

Further, we speculated that the acute intake of methamphetamine, byproducing a sudden release of 5-HT (Winslow et al., 2007) and increasedserotonergic transmission due to enhanced firing of serotonergic neuronsin raphe nuclei (Johnson, 2000; Rao et al., 2007), would result in atransient amelioration of the relative hyposerotonergic state. Becauseindividuals with this relative hyposerotonergic state might also beprone to negative affect (Young and Leyton, 2002), the relief of thesesymptoms by methamphetamine would be expected to heighten itsreinforcing effects. Consequently, there would be increased stimulustoward further methamphetamine use. Notably, however, chronicmethamphetamine use damages the terminal ends of 5-HT neurons (Nordahlet al., 2003). This decreases 5-HTT density (Sekine et al., 2006), andthe resultant disinhibition of 5-HT-mediated tonic control of dopaminerelease would serve to enhance further the rise in extracellulardopamine levels following methamphetamine use. Hypothetically, thiswould create a feed-forward pharmacological process whereby increasedmethamphetamine taking provides an ever increasing stimulus for itsfurther use.

Because this is the first study to examine whethermethamphetamine-dependent individuals who vary in genotype in the 5′HTTLPR differ in the age of onset of methamphetamine use, there are nostudies against which we can directly compare our results. Nevertheless,Johnson and colleagues (2008) showed recently that L-carriers in the 5′HTTLPR, compared with their S-carrier counterparts, have a greaterhistory and severity of lifetime drinking. However, it remains to bedetermined whether individuals who develop dependence on alcohol,methamphetamine, or both share an appreciable amount of commonlyinherited genetic traits.

This study has five notable limitations. First, this study was only apreliminary analysis with a small sample population that did not providesufficient statistical power to examine for any possible ethnic orgender differences associated with genotypic variability. Indeed, asecond caveat is that because of the small sample size, the genotypicgroups were unbalanced in size, thereby reducing our ability to drawfirm conclusions from our results. Large-scale studies are, therefore,needed to replicate and extend our findings. Third, because of thecross-sectional nature of the study, we were not able to assess howgenetic variation in the 5′ HTTLPR interacted with the progression ofmethamphetamine use over time. Fourth, our cohort was composed ofmethamphetamine-dependent individuals who were seeking treatment. Sincetreatment seekers can vary in pathophysiology from those in thecommunity, often being more motivated and generally healthier, we do notknow whether our findings can be generalized to the entire population ofthose who are using methamphetamine. Fifth, because the cohort for thisgenetic study was not drawn from the general population, but rather froma subpopulation of treatment seeking, methamphetamine-dependentindividuals, the absolute risk of an early onset of methamphetamine useamong those in the community who possess the LL genotype of 5′ HTTLPRcannot be determined.

In summary, our findings provide preliminary evidence that geneticvulnerability may be heritable, and that possession of the LL genotypein the 5′-HTTLPR might confer increased predisposition towardearly-onset methamphetamine use.

Example 8 Bibliography

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Genet. 17, 171-176.-   First, M. B., Spitzer, R. L., Gibbon, M., Williams, J. B. W., 1994.    Structured Clinical Interview for DSM-IV Axis I Disorders—Patient    Edition (SCID-I/P, Version 2.0). New York State Psychiatric    Institute, Biometrics Research Department, New York.-   Fowler, T., Lifford, K., Shelton, K., Rice, F., Thapar, A.,    Neale, M. C., McBride, A., van den Bree, M. B., 2007. Exploring the    relationship between genetic and environmental influences on    initiation and progression of substance use. Addiction 102, 413-422.-   Goldman, D., Oroszi, G., Ducci, F., 2005. The genetics of    addictions: uncovering the genes. Nat. Rev. Genet. 6, 521-532.-   Greenberg, B. D., Tolliver, T. J., Huang, S. J., Li, Q., Bengel, D.,    Murphy, D. L., 1999. Genetic variation in the serotonin transporter    promoter region affects serotonin uptake in human blood platelets.    Am. J. Med. Genet. 88, 83-87.-   Heils, A., Mossner, R., Lesch, K. P., 1997. 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P., Belmaker, R. H. (Eds.), Molecular Genetics and the    Human Personality. American Psychiatric Publishing, Washington,    D.C., pp. 109-136.-   McGue, M., Iacono, W. G., Krueger, R., 2006. The association of    early adolescent problem behavior and adult psychopathology: a    multivariate behavioral genetic perspective. Behav. Genet. 36,    591-602.-   National Institute on Drug Abuse, 2006. NIDA InfoFacts:    methamphetamine (See NIDA website). U.S. Department of Health and    Human Services, Bethesda, Md.-   Nordahl, T. E., Salo, R., Leamon, M., 2003. Neuropsychological    effects of chronic methamphetamine use on neurotransmitters and    cognition: a review. J. Neuropsychiatry Clin. Neurosci. 15, 317-325.-   Rao, H., Gillihan, S. J., Wang, J., Korczykowski, M.,    Sankoorikal, G. M., Kaercher, K. A., Brodkin, E. S., Detre, J. A.,    Farah, M. J., 2007. Genetic variation in serotonin transporter    alters resting brain function in healthy individuals. Biol.    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Example 9

The data disclosed in this example demonstrate that individuals with theTT allele have the highest craving in the human laboratory. This is ahuman laboratory cue study where individuals were presented with eitheralcohol or neutral cues. Alcoholics preferred the alcohol to the neutralcues. It can be seen below that the TT alcoholics had the highestcraving and preference for the alcohol cues compared with their Gxcounterparts. These data supplement previous studies indicating that theTT allele is associated with highest drinking severity, and that thosewith the TT allele are responsive to ondansetron treatment.

Section 1, Example 9—Difference Between Average of 4^(th) and 5^(th)Time Points and Average of 1^(st) and 2^(nd) and 3^(rd) Time Points inVisual Analog Scale (VAS) Craving

TABLE 1 Example 9: ANOVA table of difference between average of 4^(th)and 5^(th) time points and average of 1^(st) and 2^(nd) and 3^(rd) timepoints in VAS craving (TT, TG, GG) “Urge to Drink” “Crave for a Drink”Variable F-Value p-Value F-Value p-Value Treatment (Tryptophan 2.120.151 0.05 0.818 depletion) RS1042173 (TT, TG, GG) 0.23 0.796 0.10 0.901Cue 7.54 0.008 6.34 0.014 RS1042173*Cue 3.39 0.040 1.86 0.163 Age ofonset 0.61 0.441 0.26 0.613

TABLE 2 Example 9: Model-based estimates of the mean difference ofdifference between average of 4^(th) and 5^(th) time points and averageof 1^(st) and 2^(nd) and 3^(rd) time points in VAS craving (TT, TG, GG)Estimated Lower Upper VAS Comparison Difference SE p-Value 95% CI 95% CI“Urge to ALCOHOL - 7.46 2.72 0.008 2.03 12.88 drink” NEUTRAL CUE UnderTT 17.70 4.98 0.0007 7.75 27.66 ALCOHOL - NEUTRAL CUE “Crave forALCOHOL - 7.12 2.83 0.014 1.48 12.76 a drink” NEUTRAL CUE Under TT 14.985.23 0.006 4.55 25.42 ALCOHOL - NEUTRAL CUE

TABLE 3 Example 9: ANOVA table of difference between average of 4^(th)and 5^(th) time points and average of 1^(st) and 2^(nd) and 3^(rd) timepoints in VAS craving (TT, TG/GG) “Urge to Drink” “Crave for a Drink”Variable F-Value p-Value F-Value p-Value Treatment (Tryptophan 2.250.139 0.06 0.807 depletion) RS1042173 (TT, TG/GG) 0.28 0.603 0.20 0.656Cue 11.63 0.001 8.68 0.004 RS1042173*Cue 6.69 0.012 3.70 0.059 Age ofonset 0.62 0.440 0.28 0.600

TABLE 4 Model-based estimates of the mean difference of differencebetween average of 4^(th) and 5^(th) time points and average of 1^(st)and 2^(nd) and 3^(rd) time points in YAS craving (TT, TG/GG) EstimatedLower Upper VAS Comparison Difference SE p-Value 95% CI 95% CI “Urge toALCOHOL - 10.07 2.95 0.001 4.17 15.97 drink” NEUTRAL CUE Under TT 17.704.96 0.0007 7.79 27.61 ALCOHOL - NEUTRAL CUE “Crave for ALCOHOL - 9.083.08 0.004 2.93 15.23 a drink” NEUTRAL CUE Under TT 15.00 5.19 0.0054.63 25.36 ALCOHOL - NEUTRAL CUE

Section II—Example 9—Difference Between 4^(th) and 3^(rd) Time Points inVAS Craving

TABLE 1 Section II- Example 9: ANOVA table of difference between 4^(th)and 3^(rd) time points in VAS craving (TT, TG, GG) “Urge to Drink”“Crave for a Drink” Variable F-Value p-Value F-Value p-Value Treatment(Tryptophan 1.51 0.222 0.15 0.697 depletion) RS1042173 (TT, TG, GG) 0.060.942 0.14 0.866 Cue 5.71 0.019 3.30 0.073 RS1042173*Cue 2.56 0.083 2.450.093 Age of onset 0.45 0.508 0.14 0.708

TABLE 2 Section II- Example 9: Model-based estimates of the meandifference between 4^(th) and 3^(rd) time points in VAS cravingEstimated Lower Upper VAS Comparison Difference SE p-Value 95% CI 95% CI“Urge to ALCOHOL - 8.56 3.58 0.019 1.44 15.69 drink” NEUTRAL CUE UnderTT 20.33 6.61 0.003 7.18 33.49 ALCOHOL - NEUTRAL CUE “Crave forALCOHOL - 7.23 3.98 0.073 −0.70 15.16 a drink” NEUTRAL CUE Under TT19.00 7.39 0.012 4.27 33.73 ALCOHOL - NEUTRAL CUE

TABLE 3 Section II- Example 9: ANOVA table of difference between 4^(th)and 3^(rd) time points in VAS craving (TT, TG/GG) “Urge to Drink” “Cravefor a Drink” Variable F-Value p-Value F-Value p-Value Treatment(Tryptophan 1.58 0.212 0.12 0.726 depletion) RS1042173 (TT, TG/GG) 0.080.783 0.01 0.905 Cue 8.83 0.004 5.50 0.022 RS1042173*Cue 5.11 0.026 4.020.049 Age of onset 0.45 0.506 0.13 0.721

TABLE 4 Section II- Example 9: Model-based estimates of the meandifference between 4^(th) and 3^(rd) time points in VAS craving (TT,TG/GG) Estimated Lower Upper VAS Comparison Difference SE p-Value 95% CI95% CI “Urge to ALCOHOL - 11.59 3.90 0.004 3.84 19.35 drink” NEUTRAL CUEUnder TT 20.40 6.57 0.003 7.33 33.46 ALCOHOL - NEUTRAL CUE “Crave forALCOHOL - 10.25 4.37 0.022 1.54 18.95 a drink” NEUTRAL CUE Under TT18.99 7.38 0.012 4.29 33.69 ALCOHOL - NEUTRAL CUE

Example 10—Genes Associated with Topiramate Effects

Genes Associated with Efficacy of Topiramate:

1. DISC1 (Disrupted in schizophrenia gene 1)2. interactors in DISC1 pathway:

-   -   a. NDE1    -   b. PDE4D    -   c. NDEL1    -   d. PDE4B        3. Drug target receptor genes:    -   a. Kinate receptor genes: GRIKI    -   b. AMPA receptor genes: GluR1    -   c. GABA genes: GABRA1    -   d. Sodium channel proteins: SCN1A (sodium channel,        voltage-gated, type I, alpha subunit)        4. Genes associated with Topiramate metabolism and availability:    -   a. CYP2C19    -   b. CYP3A4    -   c. UGT2B7 (UDP glucuronosyltransferase 2 family, polypeptide B7)    -   d. FABP2 (fatty acid binding protein 2, intestinal)        5. Genes associated with drug resistance:    -   a. ABCB1 (ATP-BINDING CASSETTE, SUBFAMILY B, MEMBER 1)    -   b. SCN2A (voltage-gated, type II, alpha)    -   c. SCN2A2 (sodium channel, voltage-gated, type II, alpha 2        polypeptide)

Genes Associated with Adverse Effects of Topiramate:

In addition to the genes listed above, following genes have also beenstudied in association with Topiramate adverse effects:

1. Genes coding for carbonic anhydrase (CA) enzyme:

-   -   a. CA Class 11: CA2    -   b. CA Class 1V: CA4        2. Weight loss/Anorexia: ADIPOQ (adiponectin, ClQ and collagen        domain containing), HTR2C

Example 11-Analysis of Gene Patterns Related to Response to OndansetronTreatment

Genotype data was collected from 281 patients participating in aclinical trial wherein ondansetron was administered. Table 1 presentsthe coding of the raw data used for analysis: eight different genotypeswere associated with each person, for a total of N=281 subjects.

TABLE 1 Coding of Genotypes for eight genetic variants ID A B C D E F GH 100 LS TG AG AA GG CC CT AG 104 LL TG GG GG AA CC CT AA 106 LS GG GGAA GG CC CT AG 110 LS TT GG AA GG CC TT AA 113 LL TG GG AG GG CC CT GG114 SS GG GG AA GG CC CT AG 117 LS TT GG AA GG CC CC GG 119 LL TT GG GGAA CC CT AG 120 LL TT GG AA AG CC TT AA 122 LS TG GG AA AA CC TT AAGenotypes: A = 5′-HTTLPR (sert) B = rs1042173 C = rs1150226 D =rs1176713 E = rs1176719 F = rs17614942 G = rs4938056 H = rs1672717

Table 2 provides an outcome matrix wherein each person (identified by IDnumber) was assigned a responder status in several ways, ranging fromreduction of average drinks per day by 2 or more drinks (Resp.1 in Table2) to no heavy drinking days in the past 2 months (Resp.2), no more than1 heavy drinking day (Resp.3), . . . , no more than 5 heavy drinkingdays (Resp. 6).

TABLE 2 Outcome Metrics ID Resp. 1 Resp. 2 Resp. 3 Resp. 4 Resp. 5 Resp.5 Resp. 6 100 0 0 0 0 0 0 0 104 1 0 0 0 0 0 0 106 1 0 0 0 0 0 0 110 1 11 1 1 1 1 113 0 0 0 0 0 0 0 114 0 0 0 0 0 0 0 117 1 0 0 0 0 0 0 119 1 00 0 1 1 1 120 1 0 1 1 1 1 1 122 0 0 0 0 0 0 0

Because Resp.2 to Resp.6 are conceptually similar, the two outcomemeasures Resp.1 and Resp.6 were used for further analysis.

Initially, frequency tables of the data from Table 1 and otherfrequency-based on ANOVA-based analyses were performed, leading to nosignificant discrimination between responders/non-responders totreatment for any of the possible response variables. This negativeresult prompted further investigation involving:

-   -   a. Recoding of the data into binary format, which resulted in        each person being described by a binary vector of length 24;    -   b. Defining measures of association between these vectors and        the outcome from the study, e.g. a distance between the binary        vectors and the outcome measures;    -   c. Development of a search algorithm which maximized the        distances between responders and non-responders to treatment.

Certain patterns achieve good results with outcome measures Resp.1 andResp.6 and could therefore be candidates for further examination. Beloware results that illustrate this analytical concept and the construct ofpossible associations:

TABLE 3 Recoding of Gene Identifiers Identifier Expression Coded as sertLL g1LL LS g1LS SS g1SS rs1042173 TT g2TT TG g2TG GG g2GG rs1150226 AAg3AA AG g3AG GG g3GG rs1176713 AA g4AA AG g4AG GG g4GG rs1176719 AA g5AAAG g5AG GG g5GG rs17614942 AA g6AA AC g6AC CC g6CC rs4938056 CC g7CC CTg7CT TT g7TT rs1672717 AA g8AA AG g8AG GG g8GG

Each of the new variables g1LL, g1LS, . . . , g8GG can be zero or onedepending on the genotypes in the original data. For example, forsubject 100, sert is in position “LS.” This will therefore be coded asg1LL=0, g1LS=1, and g1SS=0. As a result, each subject will berepresented by a vector with length 24 and binary elements, zeroindicating “no expression” and 1 indicating “expression” of each geneposition. This way, the generally qualitative information in Table 1 istranslated in quantitative information that is suitable for furtheranalyses based on distance and association measures. To illustrate thistranslation, Table 4 presents the recoded data for the first 10subjects.

TABLE 4 Recoded Gene Expression Data: ID g1LL g1LS g1SS g2TT g2TG g2GGg3AA G3AG g3GG g4AA g4AG g4GG 100 0 1 0 0 1 0 0 1 0 1 0 0 104 1 0 0 0 10 0 0 1 0 0 1 106 0 1 0 0 0 1 0 0 1 1 0 0 110 0 1 0 1 0 0 0 0 1 1 0 0113 1 0 0 0 1 0 0 0 1 0 1 0 114 0 0 1 0 0 1 0 0 1 1 0 0 117 0 1 0 1 0 00 0 1 1 0 0 119 1 0 0 1 0 0 0 0 1 0 0 1 120 1 0 0 1 0 0 0 0 1 1 0 0 1220 1 0 0 1 0 0 0 1 1 0 0 ID g5AA g5AG g5GG g6AA g6AC g6CC g7CC G7CT g7TTg8AA g8AG g8GG 100 0 0 1 0 0 1 0 1 0 0 1 0 104 1 0 0 0 0 1 0 1 0 1 0 0106 0 0 1 0 0 1 0 1 0 0 1 0 110 0 0 1 0 0 1 0 0 1 1 0 0 113 0 0 1 0 0 10 1 0 0 0 1 114 0 0 1 0 0 1 0 1 0 0 1 0 117 0 0 1 0 0 1 1 0 0 0 0 1 1191 0 0 0 0 1 0 1 0 0 1 0 120 0 1 0 0 0 1 0 0 1 1 0 0 122 1 0 0 0 0 1 0 01 1 0 0

Association (Distance) Between Gene Expression Combinations and Outcome:

Each combination of gene expressions in Table 4 can be assigned a scorerelated to a gene pattern, depending on the number of genes from thepattern that are present in that combination. For example, if we arelooking at the pattern (g1LL, g2GG, g3AG, g7CT), which has length 4,then Subject 100 would have a score of 0+0+1+1=2 because g1LL and g2GGare in position “0” for that subject and the other two gene expressionsare in position “1”.

It follows that along this pattern (g1LL, g2GG, g3AG, g7CT) each personcan have a score ranging from 0 (no matches) to 4 (perfect match). Sucha Score can be then associated with non-responder-responder coded as 0or 1 using one of several methods for association. Here we illustratethis concept using correlations and Chi-square measures of associationand the outcome metric Resp. 1. It becomes intuitively clear that astatistically significant association between a gene pattern and theoutcome would imply ability to separate treatment responders fromnon-responders using their genotype.

Search Algorithm and Initial Results:

First, for all patterns with length of up to 4 elements, we compute ascore for each subject as described above. For example, when looking atthe pattern (g1LL, g2GG, g3AG, g7CT), the score will be computed by thefollowing sequence of commands:

-   -   Score=0;    -   If (g1LL=1) Score=Score+1;    -   If (g2GG=1) Score=Score+1;    -   If (g3AG=1) Score=Score+1;    -   If (g7CT=1) Score=Score+1.

Further, the algorithm runs through all possible patterns and computesthe association of each score with the outcome using appropriate measureof association. For example, if we use correlation as a measure ofassociation and the outcome metric Resp. 1, we will find a number ofstatistically significant associations between the Scores and theoutcome.

The best pattern is represented by: g2TT=1; g3AA=1 or g3AG=1; g4AG=1,g8AG=1

The score computed from this pattern using the equations:

Score=0.

if (g2TT eq 1) score=score+1.

if (g3AA eq 1 or g3AG eq 1) score=score+1.

if (g4AG eq 1) score=score+1.

if (g8AG eq 1) score=score+1.

is significantly correlated with the outcome Resp. 1 (r=0.23, p<0.0001)and yields the following association with responders/non responders.

TABLE 5 The best gene pattern associated with Resp. 1 Count RESPOND ColPct SCORE 0.00 1.00 2.00 3.00 4.00 Row Total 0 29 43 19 5 96 52.7 36.124.1 18.6 34.2 1 26 76 60 22 1 185 47.3 63.9 75.9 81.5 100.0 65.8 Column55 119 79 27 1 281 Total 19.6 42.3 28.1 9.6 0.4 100.0 Chi- Value DFSignificance Square 15.68157 4 0.00348

This means that with a person with Score of 0 has a 43% chance ofresponding to treatment, while a person with Score 3 or 4 (compositeresult) has over 80% chance responding to treatment (in terms of Resp.1, which is reduction of drinks/day by two or more). This association issignificant, p=0.003. It is, however, evident that if the outcome metricis changed, the strength of these associations will change as well,meaning that different gene patterns would be identified if the outcomemetric is changed.

Additional Results:

The outcome measure, i.e., the definition of “responder”, was changed toResp. 6, meaning that a person is a responder to treatment if s/he had 5or less heavy drinking days in the two months following treatment. Thisresulted in 33% of the study participants identified as responders (asopposed to 66% of the participants identified as responders by metricResp. 1). Nevertheless, a number of gene expression patterns wereidentified that favor response to treatment.

Looking at the overall results, we found that the gene expressions mostfrequently associated with non-response to treatment are: g1LL=0 (e.g.,sert in position LS or SS); g2TT=0 (e.g., rs1042173 in position TG orGG); g3GG=1 (e.g., rs1176713 in position GG); g5AA=1 (e.g., rs1176719 inposition AA), and g8GG=1 (e.g., rs1672717 in position GG). Thisobservation yields a score that is significantly associated with eitherdefinition of responder (Resp.1 or Resp.6) comprised by these genes andcomputed as follows:

-   -   score=0.    -   if (g1LS eq 1 or g1SS eq 1) score=score+1.    -   if (g2TG eq 1 or G2GG eq 1) score=score+1.    -   if (g3GG eq 1) score=score+1.    -   if (g5AA eq 1) score=score+1.    -   if (g8GG eq 1) score=score+1.        The correlations of this Score with Resp.1 and Resp. 6 are        r=−0.17 (p=0.004) and r=−0.19 (p=0.001), respectively, i.e. a        higher Score favors non-responders. This is also evident from        these cross-tables:

TABLE 6 Association of Score with Resp. 1 Count RESP1 Col Pct SCORE 0.001.00 2.00 3.00 4.00 Row Total 0 2 11 23 49 11 96 16.7 24.4 29.1 39.850.0 34.2 1 10 34 56 74 11 185 83.3 75.6 70.9 60.2 50.0 65.8 Column 1245 79 123 22 281 Total 4.3 16.0 28.1 43.8 7.4 100.0 Chi- Value DFSignificance Square 8.63234 4 0.07098 Pearson

TABLE 7 Association of Score with Resp. 6: Count RESP6 Col Pct SCORE0.00 1.00 2.00 3.00 4.00 Row Total 0 4 26 50 92 16 188 33.3 57.8 63.374.8 72.7 66.9 1 8 19 29 31 6 93 66.7 42.2 36.7 25.2 27.3 33.1 Column 1245 79 123 22 281 Total 4.3 16.0 28.1 43.8 7.4 100.0 Chi- Value DFSignificance Square 12.06335 4 0.01689 Pearson

According to Table 6, a person with a Score of 0 (composite result) hasover 80% chance to be a responder with respect to measure Resp.1 (i.e.to reduce his/her drinking by 2 or more drinks/day). This chance goesdown to 50% with a score of 4 (composite score/result). According toTable 7, a person with a Score of 0 has a 67% chance to be a responderwith respect to measure Resp.6 (i.e., to reduce his/her drinking tobelow 5 heavy drinking days in two months). This chance goes down to 27%with a score of 4.

A simple final recoding of Score (0 1=2) (2=1) (3 4=0) yields orderlycross tables showing significant associations between the increasinglevels of this variable and the likelihood for responding to treatment.For example, Table 8 presents the likelihood for Resp. 6=1 increasingfrom 25 to nearly 50% across the three categories defined by score.

TABLE 8 Final Association between Genotype and Response to treatment(defined by Resp. 6): Count Col Pct RESP6 0.00 1.00 2.00 Row Total SCORE0 108 50 30 188 74.5 63.3 52.6 66.9 1 37 29 27 93 25.5 36.7 47.4 33.1Column 145 79 57 281 Total 51.6 28.1 20.3 100.0 Value DF SignificanceChi-Square 9.47073 2 0.00878

Example 12-Additional Analysis of Gene Patterns Related to Response toOndansetron Treatment

Using the same data as was used in Example 11, further epistaticanalysis among SNPs from serotonin (SERT), 5HT-3A, and 5HT-3B revealthat significant epistatic effect exists among the three genes inaffecting response to ondesetron treatment as measured bydrinks/drinking day. Of these significant SNP combinations, the SNPcombinations of 5HTTLPR-rs1176719-rs1672717-rs2276307 and5HTTLPR-rs1042173-rs10160548-rs1176746-rs12270070 appear to be the bestto predict treatment response (see Table 1 for details).

TABLE 1 Comparison of Best Multigene Models, Prediction Accuracies,Cross-Validation Consistencies, and P Values Identified by GMDR for theassociation with drinks/drinking day Cross Validation Best SNPcombination Consistency No. of under Additive Genetic Prediction (pvalue from Loci models Accuracy sign test) P^(a) 1 rs1672717⁴(HTR3B)0.6053  7 (0.172) 0.029 2 rs1176719² (HTR3A)- 0.699 10 (0.001) <0.0001rs1672717⁴ 3 rs1176719²- rs1672717⁴- 0.6974 10 (0.001) <0.0001rs4938056(HTR3B) and 5HTTLPR¹(SERT)- 0.6867  9 (0.011) 0.002rs1176719²-rs4938056 4 5HTTLPR¹- rs1176719²- 0.702 10 (0.001) <0.0001rs1672717⁴ (HTR3B)- rs2276307⁵(HTR3B) 5 5HTTLPR¹- 0.6671  9 (0.011)0.002 rs1042173⁵(SERT)- rs10160548⁶(HTR3A)- rs1176746⁷(HTR3B)-rsl2270070⁸(HTR3B) P^(a) values from permutation test ¹LL genotype.²AG/GG genotypes. ³AA genotype. ⁴GG/AG genotypes. ⁵TT genotype. ⁶GT/GGgenotypes. ⁷GA/GG genotypes. ⁸GG genotypes.

Example 13—Ondansetron Response by Genotype: Alcohol DependentIndividuals that are T+ Carriers, in Particular Those that have the TGGenotype, of the 3′-UTR Respond Differentially to Ondansetron VersusPlacebo Treatment

Studies on 283 subjects that were randomized and evaluated according tothe 5′-HTTLPR (LL/LS/SS) and 3′-UTR (TT/TG/SS) combination wereundertaken in combination with an analysis of the pattern of response totreatment with ondansetron, in particular, whether ondansetron was moreefficacious than placebo according to genotype.

The polymorphism in the untranslated region of the serotonin transportergene (i.e., 3′UTR of SLC6A4) has been identified and determined tomodulate mRNA expression level of the serotonin transporter and isassociated with excessive drinking (Seneviratne C. et al. AlcoholClinical and Experimental Research 33(2); 332-339, 2009). Specifically,allelic differences at the rs1042173 SNP showed a significant differencein the intensity of drinking. In expression studies, the T-allele wasassociated with lower mRNA expression whilst the G-allele was associatedwith higher levels of mRNA expression. Possession of the T-allele alsowas associated with greater drinking intensity. Furthermore, it wouldappear from unpublished data that the TT genotype compared with the Gx(TG/GG) genotype was associated with the highest drinking levels. In anunpublished analysis of data from 32 non-treatment seeking alcoholics,it was observed that the TT allele compared with the Gx is associatedwith increased subjective (“Urge to drink”—F=5.58, p=0.021; “Crave for adrink”—F=5.01, p=0.028) and physiological craving for alcohol.

The 5′HTTLR and 3′-UTR regions of SLC6A4 are not linked. Therefore, itwould be surprising and unexpected for there to be any interactionbetween their polymorphisms. And certainly, even more so for there to bean interaction between these interactions that affect the consumption ofalcohol or for these alleles to predict the effect of any putativetreatment medication (including 5-HT3 antagonists) in the treatment ofalcoholism.

It is, therefore, also unexpected that from unpublished data collectedfrom a phase II study in alcohol dependent patients that there is apharmacological interaction between the T-allele and 5′-HTTLPRgenotypes. In particular, there appears to be an unexpected largetherapeutic effect to improve drinking outcomes when ondansetron isprovided to those with LL genotype who also have the TT genotype.Furthermore, and also unexpected, possession of the TG genotype adds totherapeutic effect of ondansetron on all 5′HTTLPR genotypes (i.e.,LL/LS/GG) (responders to treatment can be defined, for example, as thosefor whom the direction of effect is better for ondansetron compared withplacebo on one, two, three or four measures of response: 1) percentageof heavy drinking days (PHDD); 2) Drinks/Drinking Day (DDD); 3)Percentage of Days Abstinent (PDA); and/or 4 Percentage of patients withno heavy drinking). From the data, it was determined that LL/TT, LL/TG,LL/GG, LS/TG, and SS/TG genotypes respond to ondansetron treatment.

The TT and TG genotype groups can be summarized as T carriers. FIGS.11A-11C provide data demonstrating that LL/T+ carriers have an effect onpercent heavy drinking days, drinks/drinking days and percent daysabstinent, while FIG. 12 depicts data regarding patients with less than3 (1/month) heavy drinking days (“safe drinking”) during 12 weeks. Inconclusion, LL/T+ carriers (e.g., LL/TT and LL/TG) are responsive totreatment.

Example 14-Additional Analysis of Gene Patterns Related to Response toOndansetron Treatment

Using the same data as was used in Example 11, further epistaticanalysis among SNPs from serotonin (SERT), 5HT-3A, and 5HT-3B revealthat significant epistatic effect exists among the three genes inaffecting response to ondesetron treatment as measured bydrinks/drinking day (DDD), drinks/day (DD), percentage heavy drinkingdays (PHDD), and percentage of days abstinent (PDA).

Part I:

TABLE 1 Any one or two or three or four of 5-HTTLPR (LL) or RS1042173(TT) or RS1150226 (AG) or RS17614942 (AC) Estimate StdErr P-value 1. Oneor Two or Three or Four 5-HTTLPR (LL): −1.41 0.59 0.017 OND (n = 49) vs.Placebo (n = 44) RS1042173 (TT): −0.86 0.61 0.156 OND (n = 42) vs.Placebo (n = 48) RS1150226 (AG): −1.81 0.87 0.036 OND (n = 20) vs.Placebo (n = 24) RS17614942 (AC): −2.73 0.95 0.004 OND (n = 17) vs.Placebo (n = 19) LL + TT: −2.08 0.85 0.014 OND (n = 22) vs. Placebo (n =23) LL + AG: −3.06 1.42 0.031 OND (n = 8) vs. Placebo (n = 9) LL + AC:−4.24 1.48 0.004 OND (n = 7) vs. Placebo (n = 8) TT + AG: −2.00 1.530.191 OND (n = 9) vs. Placebo (n = 6) TT + AC: −3.37 1.44 0.019 OND (n =9) vs. Placebo (n = 7) AG + AC: −2.42 1.05 0.021 OND (n = 14) vs.Placebo (n = 16) LL + TT + AG: −3.92 2.13 0.066 OND (n = 5) vs. Placebo(n = 3) LL + TT + AC: −4.25 1.85 0.021 OND (n = 6) vs. Placebo (n = 4)LL + AG + AC: −4.09 1.69 0.016 OND (n = 5) vs. Placebo (n = 7) TT + AG +AC: −2.24 1.71 0.190 OND (n = 7) vs. Placebo (n = 5) LL + TT + AG +AC:−4.05 2.22 0.068 OND (n = 4) vs. Placebo (n = 3) 2. Any of One or Two LLor TT: −0.87 0.49 0.078 OND (n = 67) vs. Placebo (n = 68) LL or AG:−1.37 0.52 0.009 OND (n = 59) vs. Placebo (n = 58) LL or AC: −1.53 0.540.005 OND (n = 57) vs. Placebo (n = 54) TT or AG: −1.02 0.53 0.054 OND(n = 53) vs. Placebo (n = 66) TT or AC: −1.07 0.55 0.052 OND (n = 50)vs. Placebo (n = 60) AG or AC: −2.12 0.81 0.009 OND (n = 23) vs. Placebo(n = 27) 3. Any of One, Two, or Three LL or TT or AG: −0.94 0.46 0.041OND (n = 75) vs. Placebo (n = 80) LL or TT or AC: −0.97 0.47 0.038 OND(n = 74) vs. Placebo (n = 76) LL or AG or AC: −1.43 0.52 0.006 OND (n =60) vs. Placebo (60) TT or AG or AC: −0.99 0.53 0.059 OND (n = 54) vs.Placebo (n = 67) 4. Any of One, Two, Three, or Four (N = 157) LL or TTor AG or AC: −0.93 0.46 0.042 OND (n = 76) vs. Placebo (n = 81)

Table 1 Summary:

1): Except RS1042173, if patients had either 5-HTTLPR (LL) or RS1150226(AG) or RS17614942 (AC), patients who received OND had at least 1.4drinks per drinking days (DDD) reductions compared to those who receivedplacebo. If patients had either two of the four gene variants, therewere at least 2 DDD reductions with OND. If patients had either three ofthe four gene variants, there were more than 2 DDD reductions with OND.If patients had all four gene variants, there were more than 4 DDDreductions with OND. The DDD reductions seemed to increase while numberof ‘positive’ gene variants increase.

2): If patients had either one or two of the four gene variants, therewere about 0.9 to 2.1 DDD reductions with OND.

3): If patients had either one or two or three of the four genevariants, there were about 0.9 to 1.4 DDD reductions with OND.

4): If patients had either one or two or three or four of the four genevariants, there were at least 0.9 DDD reductions with OND.

Therefore, there were 157 out of 273 (56%) subjects in this study samplewho had either one or two or three or four of the four gene variantsseemed to respond to OND treatment.

TABLE 2 Any one or two or three or four of 5-HTTLPR (LL) or RS1042173(TT) or RS1176719 (AA) or RS17614942 (AC) Estimate StdErr P-value 1. Oneor Two or Three or Four 5-HTTLPR (LL): −1.41 0.59 0.017 OND (n = 49) vs.Placebo (n = 44) RS1042173 (TT): −0.86 0.61 0.156 OND (n = 42) vs.Placebo (n = 48) RSI 176719 (AA): −3.27 1.26 0.010 OND (n = 11) vs.Placebo (n = 11) RS17614942 (AC): −2.73 0.95 0.004 OND (n = 17) vs.Placebo (n = 19) LL + TT: −2.08 0.85 0.014 OND (n = 22) vs. Placebo (n =23) LL + AA: −5.22 1.82 0.004 OND (n = 7) vs. Placebo (n = 8) LL + AC:−4.24 1.48 0.004 OND (n = 7) vs. Placebo (n = 8) TT + AA: −7.12 2.10 0.0007 OND (n = 4) vs. Placebo (n = 5) TT + AC: −3.37 1.44 0.019 OND (n= 9) vs. Placebo (n = 7) AA + AC: NA NA NA OND (n = 0) vs. Placebo (n =2) LL + TT + AA: −8.50 2.32  0.0002 OND (n = 4) vs. Placebo (n = 3) LL +TT + AC: −4.25 1.85 0.021 OND (n = 6) vs. Placebo (n = 4) LL + AA + AC:−5.96 2.01 0.003 OND (n = 7) vs. Placebo (n = 3) TT + AA + AC: NA NA NAOND (n = 0) vs. Placebo (n = 1) LL + TT + AA +AC: NA NA NA OND (n = 0)vs. Placebo (n = 1) 2. Any of One or Two LL or TT: −0.87 0.49 0.078 OND(n = 67) vs. Placebo (n = 68) LI. or AA: NA NA NA OND (n = 0) vs.Placebo (n = 0) LL or AC: −1.53 0.54 0.005 OND (n = 57) vs. Placebo (n =54) TT or AA: NA NA NA OND (n = 0) vs. Placebo (n = 0) TT or AC: −1.070.55 0.052 OND (n = 50) vs. Placebo (n = 60) AA or AC: −2.83 0.76 0.0002 OND (n = 28) vs. Placebo (n = 28) 3. Any of One, Two, or ThreeLL or TT or AA: −0.99 0.48 0.039 OND (n = 70) vs. Placebo (n = 73) LL orTT or AC: −0.97 0.47 0.038 OND (n = 74) vs. Placebo (n = 76) LL or AA orAC: −1.55 0.52 0.003 OND (n = 60) vs. Placebo (n = 60) TT or AA or AC:−1.08 0.52 0.039 OND (n = 56) vs. Placebo (n = 66) 4. Any of One, Two,Three, or Four (N = 166) LL or TT or AA or AC: −1.02 0.46 0.026 OND (n =57) vs. Placebo (n = 59)

Table 2 Summary: If patients had either one or two or three or four of5-HTTLPR (LL) or RS1042173 (TT) or RS1176719 (AA) or RS17614942 (AC),they are likely to respond to OND treatment.

Part II:

TABLE 3 Any one or two or three of 5-HTTLPR (LL) or RS1042173 (TT) orRS1150226 (AG) Estimate StdErr P-value 1. One or Two or Three 5-HTTLPR(LL): −1.41 0.59 0.017 OND (n = 49) vs. Placebo (n = 44) RS1042173 (TT):−0.86 0.61 0.156 OND (n = 42) vs. Placebo (n = 48) RS1150226 (AG): −1.810.87 0.036 OND (n = 20) vs. Placebo (n = 24) LL + TT: −2.08 0.85 0.014OND (n = C2) vs. Placebo (n = 23) LL + AG: −3.06 1.42 0.031 OND (n = 8)vs. Placebo (n = 9) TT + AG: −2.00 1.53 0.191 OND (n = 9) vs. Placebo (n= 6) LL + TT + AG: −3.92 2.13 0.066 OND (n = 5) vs. Placebo (n = 3) 2.Any of One or Two LL or TT: −0.87 0.49 0.078 OND (n = 67) vs. Placebo (n= 68) LL or AG: −1.37 0.52 0.009 OND (n = 59) vs. Placebo (n = 58) TT orAG: −1.02 0.53 0.054 OND (n = 53) vs. Placebo (n = 66) 3. Any of One,Two, or Three (N = 155) LL or TT or AG: −0.94 0.46 0.041 OND (n = 75)vs. Placebo (n = 80)

TABLE 4 Any one or two or three of 5-HTTLPR (LL) or RS1042173 (TT) orRS17614942 (AC) Estimate StdErr P-value 1. One or Two or Three 5-HTTLPR(LL): −1.41 0.59 0.017 OND (n = 49) vs. Placebo (n = 44) RS1042173 (TT):−0.86 0.61 0.156 OND (n = 42) vs. Placebo (n = 48) RS17614942 (AC):−2.73 0.95 0.004 OND (n = 17) vs. Placebo (n = 19) LL + TT: −2.08 0.850.014 OND (n = 22) vs. Placebo (n = 23) LL + AC: −4.24 1.48 0.004 OND (n= 7) vs. Placebo (n = 8) TT + AC: −3.37 1.44 0.019 OND (n = 9) vs.Placebo (n = 7) LL + TT + AC: −4.25 1.85 0.021 OND (n = 6) vs. Placebo(n = 4) 2. Any of One or Two LL or TT: −0.87 0.49 0.078 OND (n = 67) vs.Placebo (n = 68) LL or AC: −1.53 0.54 0.005 OND (n = 57) vs. Placebo (n= 54) TT or AC: −1.07 0.55 0.052 OND (n = 50) vs. Placebo (n = 60) 3.Any of One, Two, or Three (N = 150) LL or TT or AC: −0.97 0.47 0.038 OND(n = 74) vs. Placebo (n = 76)

TABLE 5 Any one or two or three of RS1042173 (TT) or RS1150226 (AG) orRS17614942 (AC) Estimate StdErr P-value 1. One or Two or Three RS1042173(TT): −0.86 0.61 0.156 OND (n = 42) vs. Placebo (n = 48) RS1150226 (AG):−1.81 0.87 0.036 OND (n = 20) vs. Placebo (n = 24) RS17614942 (AC):−2.73 0.95 0.004 OND (n = 17) vs. Placebo (n = 19) TT + AG: −2.00 1.530.191 OND (n = 9) vs. Placebo (n = 6) TT + AC: −3.37 1.44 0.019 OND (n =9) vs. Placebo (n = 7) AG + AC: −2.42 1.05 0.021 OND (n = 14) vs.Placebo (n = 16) TT + AG + AC: −2.24 1.71 0.190 OND (n = 7) vs. Placebo(n = 5) 2. Any of One or Two TT or AG: −1.02 0.53 0.054 OND (n = 53) vs.Placebo (n = 66) TT or AC: −1.07 0.55 0.052 OND (n = 50) vs. Placebo (n= 60) AG or AC: −2.12 0.81 0.009 OND (n = 23) vs. Placebo (n = 27) 3.Any of One, Two, or Three (N = 121) TT or AG or AC: −0.99 0.53 0.059 OND(n = 54) vs. Placebo (n = 67)

TABLE 6 Any one or two or three of 5-HTTLPR (LL) or RS1150226 (AG) orRS17614942 (AC) Estimate StdErr P-value 1. One or Two or Three 5-HTTLPR(LL): −1.41 0.59 0.017 OND (n = 49) vs. Placebo (n = 44) RS1150226 (AG):−1.81 0.87 0.036 OND (n = 20) vs. Placebo (n = 24) RS17614942 (AC):−2.73 0.95 0.004 OND (n = 17) vs. Placebo (n = 19) LL + AG: −3.06 1.420.031 OND (n = 8) vs. Placebo (n = 9) LL + AC: −4.24 1.48 0.004 OND (n =7) vs. Placebo (n = 8) AG + AC: −2.42 1.05 0.021 OND (n = 14) vs.Placebo (n = 16) LL + AG + AC: −4.09 1.69 0.016 OND (n = 5) vs. Placebo(n = 7) 2- Any of One or Two LL or AG: −1.37 0.52 0.009 OND (n = 59) vs.Placebo (58) LL or AC: −1.53 0.54 0.005 OND (n = 57) vs. Placebo (54) AGor AC: −2.12 0.81 0.009 OND (n = 23) vs. Placebo (27) 3. Any of One,Two, or Three (N = l20) LL or AG or AC: −1.43 0.52 0.006 OND (n = 60)vs. Placebo (60)

SUMMARY

1) If patients had any one of the three gene variants (5-HTTLPR [LL],RS1150226[AG], or RS17614942 [AC]), patients who received OND had atleast 1.4 DDD reduction compared to those patients who received placebo.If patients had any two of the three gene variants, patients whoreceived OND had at least 2.4 DDD reduction compared to those whoreceived placebo. If patients had the three gene variants, patients whoreceived OND had at least 4 DDD reduction compared to those who receivedplacebo. The DDD reductions seemed to increase while the number of‘positive’ gene variants increases.

2) If patients had one or two of the three gene variants, patients whoreceived OND had about 1.4 DDD reductions compared to those who receivedplacebo. 3): If patients had one or two or three of the three genevariants, patients who received OND had at least 1.4 DDD reductionscompared to those who received placebo.

This sub-sample, with 120 patients, had the strongest among three genevariants combinations (All p-values<0.05).

Patients who had either one or two or three of these three gene variantsseemed to respond to Ondansetron treatment.

TABLE 7 Any one or two or three of 5-HTTLPR (LL) or RS1042173 (TT) orRS1176713 (GG) Estimate StdErr P-value 1. One or Two or Three 5-HTTLPR(LL): −1.41 0.59 0.017 OND (n = 49) vs. Placebo (n-44) RS1042173 (TT):−0.86 0.61 0.156 OND (n = 42) vs. Placebo (n = 48) RS1176713 (GG): −3.921.57 0.013 OND (n = 6) vs. Placebo (n = 9) LL + TT: −2.08 0.85 0.014 OND(n = 22) vs. Placebo (n = 23) LL + GG: −4.48 1.98 0.024 OND (n = 5) vs.Placebo (n = 4) TT + GG: −6.42 2.41 0.008 OND (n = 3) vs. Placebo (n =4) LL + TT + GG: −7.65 2.55 0.003 OND (n = 3) vs. Placebo (n = 3) 2. Anyof One or Two LL or TT: −0.87 0.49 0.078 OND (n = 67) vs. Placebo (n =68) LL or GG: −1.60 0.58 0.006 OND (n = 47) vs. Placebo (n = 48) IT orGG: −1.02 0.59 0.082 OND (n = 45) vs. Placebo (n = 53) 3. Any of One.Two, or Three (N = 139) LL or TT or GG: −0.94 0.46 0.041 OND (n = 67)vs. Placebo (n = 72)

Summary: In this section II the first four tables were really sub-sampleof the four gene variants described in the section I (5-HTTLPR (LL) orRS1042173 (TT) or RS1150226 (AG) or RS17614942 (AC)).

In table 7, except RS1042173 (TT), if patients had either 5-HTTLPR (LL)or RS1176713 (GG), they had at least 1.4 DDD reductions with OND. Ifpatients had any two of the three gene variants, they had at least 2 DDDreductions with OND. If patients had the three gene variants, they hadat least 7 DDD reductions with OND. If patients had one or two of thethree gene variants, they had about 0.9 to 1.6 DDD reductions with OND.If patients had one, or two or three of the three gene variants, theyhad at least 0.9 DDD reductions with OND. The sample size was 139.

Part III:

TABLE 8 Any one or two or three of 5-HTTLPR (LL) or RS1042173 (TT) orRS1176719 (AA) Estimate StdErr P-value 1. One or Two or Three 5-HTTLPR(LL): −1.41 0.59 0.017 OND (n = 49) vs. Placebo (n = 44) RS1042173 (TT):−0.86 0.61 0.156 OND (n = 42) vs. Placebo (n = 48) RS1176719 (AA): −3.271.26 0.010 OND (n = 11) vs. Placebo (n = 11) LL + TT: −2.08 0.85 0.014OND (n = 22) vs. Placebo (n = 23) LL + AA: −5.22 1.82 0.004 OND (n = 7)vs. Placebo (n = 8) TT + AA: −7.12 2.10 0.0007 OND (n = 4) vs. Placebo(n = 5) LL + TT + AA: −8.50 2.32 0.0002 OND (n = 4) vs. Placebo (n = 3)2, Any of One or Two LL or TT: −0.87 0.49 0.078 OND (n = 67) vs. Placebo(n = 68) LL or AA: NA NA NA OND (n = 0) vs. Placebo (n = 0) TT or AA: NANA NA OND (n = 0) vs. Placebo (n = 0) 3. Any of One,Two, or Three (N =143) LL or TT or AA: −0.99 0.48 0.039 OND (n = 70) vs. Placebo (n = 73)

TABLE 9 Any one or two or three of 5-HTTLPR (LL) or RS1176719 (AA) orRS17614942 (AC) Estimate StdErr P-value 1. One or Two or Three 5-HTTLPR(LL): −1.41 0.59 0.017 OND (n = 49) vs. Placebo (n = 44) RS1176719 (AA):−3.27 1.26 0.001 OND (n = 11) vs. Placebo (n = 11) RS17614942 (AC):−2.73 0.95 0.004 OND (n = 17) vs. Placebo (n = 19) LL + AA: −5.22 1.820.004 OND (n = 7) vs. Placebo (n = 4) LL + AC: −4.24 1.48 0.004 OND (n =7) vs. Placebo (n = 8) AA + AC: NA NA NA OND (n = 0) vs. Placebo (n = 2)LL + AA + AC: −5.96 2.01 0.003 OND (n = 7) vs. Placebo (n = 3) 2. Any ofOne or Two LL or AA: NA NA NA OND (n = 0) vs. Placebo (n = 0) LL or AC:−1.53 0.54 0.005 OND (n = 57) vs. Placebo (n = 54) AA or AC: −2.83 0.76 0.0002 OND (n = 28) vs. Placebo (n = 28) 3. Any of One, Two, or Three(N = 120) LL or AA or AC: −1.55 0.52 0.003 OND (n = 60) vs. Placebo (n =60)

TABLE 10 Any one or two or three of RS1042173 (TT) or RS1176719 (AA) orRS17614942 (AC) Estimate StdErr P-value 1. One or Two or Three RS1042173(TT): −0.86 0.61 0.156 OND (n = 42) vs. Placebo (n = 48) RS1176719 (AA):−3.27 1.26 0.010 OND (n = 11) vs. Placebo (n = 11) RS17614942 (AC):−2.73 0.95 0.004 OND (n = 17) vs. Placebo (n = 19) TT + AA: −7.12 2.100.0007 OND (n = 4) vs. Placebo (n = 5) TT + AC: −3.37 1.44 0.019 OND (n= 9) vs. Placebo (n = 7) AA + AC: NA NA NA OND (n = 0) vs. Placebo (n =2) TT + AA + AC: NA NA NA OND (n = 0) vs. Placebo (n = 1) 2. Any of Oneor Two TT or AA: NA NA NA OND (n = 0) vs. Placebo (n = 0) TT or AC:−1.07 0.55 0.052 OND (n = 50) vs. Placebo (n = 60) AA or AC: −2.83 0.760.0002 OND (n = 28) vs. Placebo (n = 28) 3. Any of One, Two, or Three (N= 122) TT or AA or AC: −1.08 0.52 0.039 OND (n = 56) vs. Placebo (n =66)

TABLE 11 Any one or two or three of 5-HTTLPR (LL) or RS1176713 (GG) orRS17614942 (AC) Estimate StdErr P-value 1. One or Two or Three 5-HTTLPR(LL): −1.41 0.59 0.017 OND (n = 49) vs. Placebo (n = 44) RS1176713 (GG):−3.92 1.57 0.013 OND (n = 6) vs. Placebo (n = 9) RS17614942 (AC): −2.730.95 0.004 OND (n = 17) vs. Placebo (n = 19) LL + GG: −4.48 1.98 0.024OND (n = 5) vs. Placebo (n = 4) LL + AC: −4.24 1.48 0.004 OND (n = 7)vs. Placebo (n = 8) GG + AC: NA NA NA OND (n = 0) vs. Placebo (n = 2)LL + GG + AC: NA NA NA OND (n = 0) vs. Placebo (n = 1) 2. Any of One orTwo LL or GG: −1.60 0.58 0.006 OND (n = 47) vs. Placebo (48) LL or AC:−1.53 0.54 0.005 OND (n = 57) vs. Placebo (54) GG or AC: −3.08 0.82 0.0002 OND (n = 23) vs. Placebo (26) 3. Any of One, Two, or Three (N =115) LL or GG or AC: −1.57 0.53 0.003 OND (n = 57) vs. Placebo (58)

TABLE 12 Any one or two or three of RS1042173 (TT) or RS1150226 (AG) orRS1176713 (GG) Estimate StdErr P-value 1. One or Two or Three RS1042173(TT): −0.86 0.61 0.156 OND (n = 42) vs. Placebo (n = 48) RS1150226 (AG):−1.81 0.87 0.036 OND (n = 20) vs. Placebo (n = 24) RS1176713 (GG): −3.921.57 0.013 OND (n = 6) vs. Placebo (n = 9) TT + AG: −2.00 1.53 0.191 OND(n = 9) vs. Placebo (n = 6) TT + GG: −6.42 2.41 0.008 OND (n = 3) vs.Placebo (n = 4) AG + GG: NA NA NA OND (n = 0) vs. Placebo (n = 0) TT +AG + GG: NA NA NA OND (n = 0) vs. Placebo (n = 0) 2. Any of One or TwoTT or AG: −1.02 0.53 0.054 OND (n = 53) vs. Placebo (n = 66) TT or GG:−1.02 0.59 0.082 OND (n = 45) vs. Placebo (n = 53) AG or GG: −2.43 0.750.001 OND (n = 26) vs. Placebo (n = 33) 3. Any of One, Two or Three (N =127) TT or AG or GG: −1.12 0.51 0.030 OND (n = 56) vs. Placebo (n = 71)

TABLE 13 Any one or two or three of RS1042173 (TT) or RS1176713 (GG) orRS17614942 (AC) Estimate StdErr P-value 1. One or Two or Three RS1042173(TT): −0.86 0.61 0.156 OND (n = 42) vs. Placebo (n = 48) RS1176713 (GG):−3.92 1.57 0.013 OND (n = 6) vs. Placebo (n = 9) RS17614942 (AC): −2.730.95 0.004 OND (n = 17) vs. Placebo (n = 19) TT + GG: −6.42 2.41 0.008OND (n = 3) vs. Placebo (n = 4) TT + AC: −3.37 1.44 0.019 OND (n = 9)vs. Placebo (n = 7) GG + AC: NA NA NA OND (n = 0) vs. Placebo (n = 2)TT + GG + AC: NA NA NA OND (n = 0) vs. Placebo (n = 1) 2. Any of One orTwo TT or GG: −1.02 0.59 0.082 OND (n = 45) vs. Placebo (n = 53) TT orAC: −1.07 0.55 0.052 OND (n = 50) vs. Placebo (n = 60) GG or AC: −3.080.82 0.076 OND (n = 23) vs. Placebo (n = 26) 3. Any of One, Two, orThree (N = 117) TT or GG or AC: −1.12 0.54 0.037 OND (n = 53) vs.Placebo (n = 64)

Additional Analysis of Gene Patterns Related to Response to OndansetronTreatment

Using the same data as was used in Example 11, further epistaticanalysis among SNPs from serotonin (SERT), 5HT-3A, and 5HT-3B revealthat significant epistatic effect exists among the three genes inaffecting response to ondesetron treatment as measured by DDD, DD, PHDD,and PDA.

TABLE 1 Multigene SNP combinations associated with AD and ondansetrontreatment outcome SNP and Alcohol Dependence (AD) OND treatment responseGenotype χ² χ² OND- OND-non- χ² χ² Response combination AD N Controls Nvalue* P value responders N responders N value P value** variablers3758987 + 5HTTLPR(5) 125 51 1.1613 0.2812 23 16 3.1065 0.078 More than3 TT/CT + LL standard all other genotype combinations 288 167 34 52drinks rs3758987 + rs1042173(7) 128 61 2.6371 0.1044 22 14 4.5637 0.0327improvement TT/CT + TT from the all other genotype combinations 285 15735 54 baseline in 5-HTTLPR + rs1042173(6) 64 34 1.1505 0.2835 16 44.9122 0.0267 the last 2 LL + TT months all other genotype combinations349 184 41 64 rs3758987 + rs2276307 + 5-HTTLPR(2) 73 33 0.004 0.9493 2012 4.1519 0.0416 TT/CT + AA + LL all other genotype combinations 340 18537 56 rs3758987 + 5-HTTLPR + rs1042173(4) 59 29 4.2689 0.0388 15 44.4385 0.0351 TT/CT + LL + TT all other genotype combinations 354 189 4264 rs3758987 + rs2276307 + 5- 35 17 1.6332 0.2013 13 2 7.3047 0.0069HTTLPR + rs1042173(3) TT/CT + AA + LL + TT all other genotypecombinations 378 201 44 66

Example 16-Additional Analysis of Gene Patterns Related to Response toOndansetron Treatment

Using the same data as was used in Example 11, further epistaticanalysis among SNPs from serotonin (SERT), 5HT-3A, and 5HT-3B revealthat significant epistatic effect exists among the three genes inaffecting response to ondesetron treatment as measured by DDD, DD, andPDA.

LL+TT or RS17614942 (AC) or RS1150226 (AG)

TABLE 1A ANOVA of Drinks per Drinking Days Drinks per Drinking DaysVariable F-Value P-Value Comb 9.91 0.002 Treatment 3.81 0.051Comb*Treatment 7.59 0.006 Comb: (LL + TT or AC or AG), and Others

TABLE 1B Least Squares Mean Effect Estimated Mean Standard Error Others6.27 0.24 Comb 5.09 0.32 Placebo 6.04 0.27 OND 5.32 0.29 Others: Placebo(n = 92) 6.12 0.31 OND (n = 97) 6.42 0.31 Comb: Placebo (n = 46) 5.970.42 OND (n = 38) 4.21 0.47

TABLE 1C Least Squares Mean Difference between Treatment and Placebo andIts 95% Confidence Intervals Estimated Mean Lower Upper EffectDifference 95% C.I. 95% C.I. P-Value Among Comb: OND vs. Placebo −1.75−2.98 −0.53 0.005 Among OND: Comb vs. Others −2.21 −3.29 −1.13 <0.0001

Note: 84 out of 273 patients in this study (31%). Among the patients whohad LL+TT or AC or AG had at least 1.75 DDD reduction compared OND withplacebo groups.

TABLE 2A ANOVA of Drinks per Days Drinks per Days Variable F-ValueP-Value Comb 3.88 0.049 Treatment 3.93 0.048 Comb*Treatment 7.77 0.005Comb: (LL + TT or AC or AG), and Others

TABLE 2B Least Squares Mean Effect Estimated Mean Standard Error Others4.41 0.22 Comb 3.72 0.30 Placebo 4.41 0.25 OND 3.72 0.27 Others: Placebo(n = 92) 4.27 0.29 OND (n = 97) 4.55 0.29 Comb: Placebo (n = 46) 4.550.39 OND (n = 38) 2.90 0.44

TABLE 2C Least Squares Mean Difference between Treatment and Placebo andIts 95% Confidence Intervals Estimated Mean Lower Upper EffectDifference 95% C.I. 95% C.I. P-Value Among Comb: OND vs. Placebo −1.65−2.78 −0.52 0.004 Among OND: Comb vs. Others −1.65 −2.64 −0.65 0.001

TABLE 3A ANOVA of Percentage Days of Abstinent PDA Variable F-ValueP-Value Comb 3.00 0.083 Treatment 3.33 0.068 Comb*Treatment 4.30 0.038Comb: (LL + TT or AC or AG), and Others

TABLE 3B Least Squares Mean Effect Estimated Mean Standard Error Others36.01 2.27 Comb 42.00 3.01 Placebo 35.89 2.58 OND 42.13 2.73 Others:Placebo (n = 92) 36.45 2.95 OND (n = 97) 35.58 2.95 Comb: Placebo (n =46) 35.32 3.94 OND (n = 38) 48.68 4.36

TABLE 3C Least Squares Mean Difference between Treatment and Placebo andIts 95% Confidence Intervals Estimated Mean Lower Upper EffectDifference 95% C.I. 95% C.I. P-Value Among Comb: OND vs. Placebo 13.362.14 24.58 0.020 Among OND: Comb vs. Others 13.11 3.18 23.03 0.010

-   -   RS17614942 is located in HTR3B intron 8

TABLE 2A Frequency of RS17614942 RS17614942 Frequency (%) True FrequencyAA 2 (0.7%) Very rare AC 36 (13.1%) 13% CC 236 (86.1%)  86%

-   -   -   a. Combined AC with AA

TABLE 2B ANOVA of Drinks per Drinking Days Drinks per Drinking DaysVariable F-Value P-Value RS17614942 (CC, AC/AA) 2.62 0.101 Treatment5.79 0.016 RS17614942*Treatment 6.92 0.009

TABLE 2C Least Squares Mean Effect Estimated Mean Standard Error AC/AA5.15 0.48 CC 5.97 0.23 Placebo 6.16 0.37 OND 4.96 0.39 AC/AA: Placebo (n= 20) 6.40 0.65 OND (n = 18) 3.90 0.69 CC: Placebo (n = 119) 5.92 0.29OND (n = 117) 6.03 0.30

TABLE 2D Least Squares Mean Difference between Treatment and Placebo andIts 95% Confidence Intervals Estimated Mean Lower Upper EffectDifference 95% C.I. 95% C.I. P-Value Among AC/AA: OND vs. Placebo −2.51−4.32 −0.70 0.007 Among OND: AC/AA vs. CC −2.13 −3.55 −0.71 0.003 ONDvs. Placebo −1.20 −2.11 −0.19 0.016

-   -   -   b. Combined AA with CC

TABLE 2E ANOVA of Drinks per Drinking Days Drinks per Drinking DaysVariable F-Value P-Value RS17614942 (CC/AA, AC) 2.43 0.120 Treatment6.55 0.011 RS17614942*Treatment 7.84 0.005

TABLE 2F Least Squares Mean Effect Estimated Mean Standard Error AC 5.230.48 CC/AA 6.03 0.22 Placebo 6.28 0.36 OND 4.98 0.38 AC: Placebo (n =19) 6.59 0.65 OND (n = 17) 3.87 0.69 CC/AA: Placebo (n = 120) 5.97 0.28OND (n = 118) 6.09 0.29

TABLE 2G Least Squares Mean Difference between Treatment and Placebo andIts 95% Confidence Intervals Estimated Mean Lower Upper EffectDifference 95% C.I. 95% C.I. P-Value Among AC: OND vs. Placebo −2.73−4.59 −0.87 0.004 Among OND: AC/AA vs. CC −2.22 −3.68 −0.77 0.003 ONDvs. Placebo −1.30 −2.30 −0.30 0.011

-   -   RS1150226 is located on ˜500 bp upstream of HTR3A gene, possibly        within HTR3A promoter

TABLE 3A RS17614942 Frequency (%) True Frequency AA 2 (0.7%) Very rareAG 44 (16.1%) 13% GG 228 (83.2%)  86%

-   -   -   a. Combined AG with AA

TABLE 3B ANOVA of Drinks per Drinking Days Drinks per Drinking DaysVariable F-Value P-Value RS1150226 (GG, AG/AA) 4.53 0.033 Treatment 3.560.059 RS1150226*Treatment 4.06 0.044

TABLE 3C Least Squares Mean Effect Estimated Mean Standard Error AG/AA5.10 0.43 GG 6.08 0.22 Placebo 6.02 0.33 OND 5.15 0.35 AG/AA: Placebo (n= 24) 6.00 0.59 OND (n = 22) 4.20 0.61 GG: Placebo (n = 115) 6.05 0.28OND (n = 13) 6.11 0.29

TABLE 3D Least Squares Mean Difference between Treatment and Placebo andIts 95% Confidence Intervals Estimated Mean Lower Upper EffectDifference 95% C.I. 95% C.I. P-Value Among AG/AA: OND vs. Placebo −1.80−3.46 −0.15 0.033 Among OND: AG/AA vs. GG −1.91 −3.22 −0.61 0.004 AG/AAvs. GG −0.98 −1.89 −0.08 0.033

-   -   -   b. Combined GG with AA

TABLE 3E ANOVA of Drinks per Drinking Days Drinks per Drinking DaysVariable F-Value P-Value RS1150226 (GG/AA, AG) 4.28 0.039 Treatment 3.580.059 RS1150226*Treatment 3.80 0.051

TABLE 3F Least Squares Mean Effect Estimated Mean Standard Error AG 6.070.22 GG/AA 5.09 0.44 Placebo 6.03 0.33 OND 5.13 0.36 AG: Placebo (n =24) 6.05 0.28 OND (n = 20) 6.08 0.29 GG/AA: Placebo (n = 115) 6.00 0.59OND (n = 115) 4.19 0.64

TABLE 3G Least Squares Mean Difference between Treatment and Placebo andIts 95% Confidence Intervals Estimated Mean Lower Upper EffectDifference 95% C.I. 95% C.I. P-Value Among AG: OND vs. Placebo −1.81−3.51 −0.12 0.036 Among OND: AG vs. GG/AA −1.90 −3.26 −0.54 0.006 AG vs.GG/AA −0.98 −1.90 −0.05 0.039

RS1150226 (AG) or RS17614942 (AC)

TABLE 4A ANOVA of Drinks per Drinking Days Drinks per Drinking DaysVariable F-Value P-Value AG or AC 3.25 0.072 Treatment 4.89 0.027 (AG orAC)*Treatrnent 6.32 0.012

TABLE 4B Least Squares Mean Effect Estimated Mean Standard Error Others6.05 0.22 AG or AC 5.25 0.41 Placebo 6.14 0.32 OND 5.16 0.34 Others:Placebo (n = 111) 5.99 0.29 OND (n = 112) 6.12 0.30 AG or AC: Placebo (n= 27) 6.30 0.55 OND (n = 23) 4.19 0.60 Others: AA/GG and AA/CC

TABLE 4C Least Squares Mean Difference between Treatment and Placebo andIts 95% Confidence Intervals Estimated Mean Lower Upper EffectDifference 95% C.I. 95% C.I. P-Value Among AG or/and AC: OND vs. Placebo−2.12 −3.70 −0.53 0.009 Among OND: (AG or AC) vs. Others −1.91 −3.22−0.61 0.004 OND vs. Placebo −0.99 −1.87 −0.11 0.027

The disclosures of each and every patent, patent application, andpublication cited herein are hereby incorporated by reference herein intheir entirety.

Headings are included herein for reference and to aid in locatingcertain sections. These headings are not intended to limit the scope ofthe concepts described therein under, and these concepts may haveapplicability in other sections throughout the entire specification.

While this invention has been disclosed with reference to specificembodiments, it is apparent that other embodiments and variations ofthis invention may be devised by others skilled in the art withoutdeparting from the true spirit and scope of the invention.

BIBLIOGRAPHY

-   1. Chen, et al., “Effects of Topiramate and Other Anti-Glutamatergic    Drugs on the Acute Intoxicating Actions of Ethanol in Mice:    Modulation by Genetic Strain and Stress”, Neuropsychopharmacology    (2009), 34, 1454-1466.-   2. Ray, et al., “A Preliminary Pharmacogenetic Investigation of    Adverse Events From Topiramate in Heavy Drinkers”, Experimental and    Clinical Psychopharmacology, 2009, Vol. 17, No. 2, 122-129.-   3. Nallani, et al., “Dose-Dependent Induction of Cytochrome P450    (CYP) 3A4 and Activation of Pregnane X Receptor by Topiramate”,    Epilepsia, 44 (12): 1521-1528. 2003.-   4. Johnson, et al., “Topiramate for Treating Alcohol Dependence: A    Randomized Controlled Trial”, JAMA, 2007; 298 (14): 1641-1651.-   5. Johnson, et al., “Oral Topiramate for Treatment of Alcohol    Dependence: A Randomised Controlled Trial”, The Lancet, Vol 361,    1677-85, 2003.

What is claimed is:
 1. A method of treating an addictive disease ordisorder, comprising: administering to a patient in need thereof atherapeutically effective amount of ondansetron, wherein the patient isknown to have a genotype selected from sets (a)-(g): a. the GG genotypeof rs1176713 of gene HTR3A; b. the GG genotype of rs1176713 of geneHTR3A and the AC genotype of rs17614942 of gene HTR3A; c. the GGgenotype of rs1176713 of gene HTR3A and the AG genotype of rs1150226 ofgene HTR3A; d. the GG genotype of rs1176713 of gene HTR3A, the ACgenotype of rs17614942 of gene HTR3A, and the AG genotype of rs1150226of gene HTR3A; e. the GG genotype of rs1176713 of gene HTR3A, the LLgenotype of 5-HTTLPR and the TT genotype of rs1042173 of gene SLC6A4; f.the GG genotype of rs1176713 of gene HTR3A, the AC genotype ofrs17614942 of gene HTR3A, the LL genotype of 5-HTTLPR, and the TTgenotype of rs1042173 of gene SLC6A4; and, g. the GG genotype ofrs1176713 of gene HTR3A, the AG genotype of rs1150226 of gene HTR3A, theLL genotype of 5-HTTLPR, and the TT genotype of rs1042173 of geneSLC6A4; wherein the addictive disease or disorder is selected from thegroup consisting of: alcohol-related diseases and disorders andopioid-related disorders.
 2. The method of claim 1, wherein the patientis known to have genotype set (a).
 3. The method of claim 1, wherein thepatient is known to have genotype set (b).
 4. The method of claim 1,wherein the patient is known to have genotype set (c).
 5. The method ofclaim 1, wherein the patient is known to have genotype set (d).
 6. Themethod of claim 1, wherein the patient is known to have genotype set(e).
 7. The method of claim 1, wherein the patient is known to havegenotype set (f).
 8. The method of claim 1, wherein the patient is knownto have genotype set (g).
 9. The method of claim 1, wherein ondansetronis administered at a dosage ranging from 0.1 μg/kg to 1000 μg/kg perapplication.
 10. The method of claim 1, wherein ondansetron isadministered at a dosage of 1 μg/kg to 30 μg/kg per application.
 11. Themethod of claim 1, wherein ondansetron is administered at a dosage of 1μg/kg per application.
 12. The method of claim 1, wherein ondansetron isadministered at a dosage of 2 μg/kg per application.
 13. The method ofclaim 1, wherein ondansetron is administered at a dosage of 3 μg/kg perapplication.
 14. The method of claim 1, wherein ondansetron isadministered at a dosage of 4 μg/kg per application.
 15. The method ofclaim 1, wherein ondansetron is administered at a dosage of 5 μg/kg perapplication.
 16. The method of claim 1, wherein ondansetron isadministered at a dosage of 6 μg/kg per application.
 17. The method ofclaim 1, wherein ondansetron is administered at a dosage of 7 μg/kg perapplication.
 18. The method of claim 1, wherein ondansetron isadministered at a dosage of 8 μg/kg per application.
 19. The method ofclaim 1, wherein ondansetron is administered at a dosage of 9 μg/kg perapplication.
 20. The method of claim 1, wherein ondansetron isadministered at a dosage of 10 μg/kg per application.
 21. The method ofclaim 1, wherein the ondansetron is administered once a day.
 22. Themethod of claim 1, wherein the ondansetron is administered twice a day.23. The method of claim 1, wherein the addictive disease or disorder isan alcohol-related disease or disorder.
 24. The method of claim 23,wherein the alcohol-related disease or disorder is selected from thegroup consisting of: early onset alcoholism, late onset alcoholism,alcohol-induced psychotic disorder with delusions, alcohol abuse, heavydrinking, excessive drinking, alcohol intoxication, alcohol withdrawal,alcohol intoxication delirium, alcohol withdrawal delirium,alcohol-induced persisting dementia, alcohol-induced persisting amnesticdisorder, alcohol dependence, alcohol-induced psychotic disorder withhallucinations, alcohol-induced mood disorder, alcohol-induced orassociated bipolar disorder, alcohol-induced or associatedpost-traumatic stress disorder, alcohol-induced anxiety disorder,alcohol-induced sexual dysfunction, alcohol-induced sleep disorder,alcohol-induced or associated gambling disorder, alcohol-induced orassociated sexual disorder, alcohol-related disorder not otherwisespecified, alcohol intoxication, and alcohol withdrawal.
 25. The methodof claim 23, wherein a response from the treatment, comprises: areduction in drinking.
 26. The method of claim 23, wherein the reductionin drinking, comprises a reduction of heavy drinking.
 27. The method ofclaim 23, wherein the reduction in drinking, comprises a reduction ofexcessive drinking.
 28. The method of claim 23, wherein the reduction indrinking, comprises a reduction of drinks/day.
 29. The method of claim23, wherein the reduction in drinking, comprises a reduction of drinksper drinking day.
 30. The method of claim 1, wherein the addictivedisease or disorder is an opioid-related disorder.
 31. The method ofclaim 28, wherein the opioid-related disorder is selected from the groupconsisting of: opioid dependence, opioid abuse, opioid intoxication,opioid intoxication delirium, opioid-induced psychotic disorder, withdelusions, opioid-induced psychotic disorder with hallucinations,opioid-induced anxiety disorder, opioid-related disorder not otherwisespecified (nos), opioid intoxication, and opioid withdrawal.